FIELD OF THE DISCLOSURE
[0001] The technology provided herein relates to methods of insect pest control by incorporating
an inhibitor against the structural sheath protein (SHP) into the body of an agricultural
target pest, and to pest control agents to be used in the method and to transgenic
crop, greenhouse and ornamental plants.
BACKGROUND
[0002] The environment in which humans live is replete with pest infestation. Pests including
insects, arachnids, crustaceans, fungi, bacteria, viruses, nematodes, flatworms, roundworms,
pinworms, hookworms, tapeworms, trypanosomes, schistosomes, botflies, fleas, ticks,
mites, and lice and the like are pervasive in the human environment. For example,
insects of the order Hemiptera including aphids are significant pests of crops and
garden plants as well as ornamentals.
[0003] In whole Europe, direct damage only by aphids is responsible for mean annual losses
of 700,000 t of wheat, 850,000 t of potatoes and 2,000,000 t of sugar beet (Wellings
et al., 1989). In the USA, annual direct yield losses in wheat and barley production,
through reduced yields and pesticide treatment, peaked at $274 million in 1988 and
dropped to less than $10 million by 1993 (Dedryver et al., 2010). In the UK direct
yield losses from aphids is 8-16% in pea, 10-13% in wheat and 5% in potato (Tatchell,
1989). In this context, virus transmission, e.g. the barley yellow dwarf virus or
potato leaf roll virus; represents an important factor.
[0004] For aphid control, chemical agents as e.g. Imidacloprid and Dimethoat are used in
conventional plant production while for biological plant production Azadirachtin from
the Neem tree is applied (http://www.profiflor.de/index.htm). A further approach in
aphid control is the use of beneficial insects (hoverfly, ladybeetle, brown lacewing)
but this approach is only suitable for greenhouse cultures and can lead to the manifestation
of invasive species. However, the use of insecticides is the most important control
mechanism for aphids but the number of accredited insecticides was reduced during
the last years due to potentially negative influences on the environment. An additional
problem with insecticides is the fact that beside a variety of other insect species,
aphids were shown to develop resistances. While the melon and cotton aphid
Aphis gossypii actually shows resistances to 41 active compounds the green peach aphid
Myzus persicae already developed resistances against 74 compounds (http://www.pesticideresistance.com/).
Insecticide resistances can already occur after one generation and were reported in
different aphid species and populations all over the world.
[0005] To prevent negative environmental effects of insecticides and to decrease the risk
of resistance development, the strategy of integrated pest management (IPS) was developed
to minimize the amount of applied pesticides (insecticides and herbicides). IPS is
for example obligate for agriculture in Germany in accordance to the "Gute fachliche
Praxis" and charges the use of biological, biotechnical and plant breeding approaches
as well as agricultural culture methods. IPS is declared by the United Nations as
general principle for plant protection.
[0006] To reduce the amount of insecticides, new biotechnical approaches are developed in
accordance with IPS to control pests in agriculture. One of these approaches is the
use of RNA interference. With regard to aphids, RNAi-mediated gene silencing was achieved
in a number of publications by injection of dsRNA or siRNAs into the hemolymph (Mutti
et al., 2006; Jaubert-Possamai et al., 2007) or artificial feeding of dsRNA (Shakesby
et al., 2009; Whyard et al., 2009).
[0007] The very first proof of concept for transgenic plants that deliver highly specific
dsRNA to their aphid hosts was conducted by Pitino et al. (2011). The authors selected
rack1 (gut located) and c002 (salivary gland located) as two different gene targets
for the green peach aphid
Myzus persicae. Two different plants,
Nicotiana benthamiana and
Arabidopsis thaliana, were transformed for each target and a silencing effect in aphids of up to 60% was
observed on respective GM plant species. As a consequence of gene silencing the authors
described for both genes a reduced fecundity. Surprisingly, silencing of C002 did
not influence survival as previously observed for
in vitro experiments with the pea aphid
Acyrthosiphon pisum (Mutti et al., 2006). The authors suggest that this discrepancy is related to the
different species.
[0008] Because most plants are infested by more than one pest species an approach is needed
whose efficiency does not differ between different species.
[0009] Therefore, the availability of improved pest control methods for numerousness pest
species would be highly advantageous.
SUMMARY OF THE DISCLOSURE
[0010] The present disclosure pertains in particular two methods of RNAi mediated silencing
of the sheath protein SHP for control of plant sucking insects of the order Hemiptera,
in particular of the groups Sternorryhncha and Fulgoromorpha in agriculture.
[0011] In a first aspect, embodiments of the disclosure provide novel pest control methods
comprising incorporating an inhibitor against the structural sheath protein (SHP)
into the body of an agricultural target pest.
[0012] In a second aspect, embodiments of this disclosure relate to isolated polynucleotides
selected from the group consisting of:
- a) a polynucleotide comprising a nucleic acid sequence selected from the group consisting
of SEQ ID NO:1, SEQ ID NO:5 and SEQ ID NO:7;
- b) a polynucleotide that hybridizes to a nucleic acid sequence selected from the group
consisting of SEQ ID NO:1, SEQ ID NO:5 and SEQ ID NO:7 under stringent conditions;
- c) a polynucleotide of at least 70, at least 80, at least 85, at least 90 percent
sequence identity, to a nucleic acid sequence selected from the group consisting of
SEQ ID NO:1, SEQ ID NO:5 and SEQ ID NO:7;
- d) a fragment of at least 16 contiguous nucleotides of a nucleic acid sequence selected
from the group consisting of SEQ ID NO:1, SEQ ID NO:5 and SEQ ID NO:7; and
- e) a complement of the sequence of (a), (b), (c) or (d),
wherein ingestion by a Hemiptera crop, greenhouse and/or ornamental plant pest of
a double stranded ribonucleotide sequence comprising at least one strand that is complementary
to said polynucleotide or said fragment reduce feeding of said pest.
[0013] In a third aspect, embodiments of this disclosure relate to double stranded ribonucleotide
sequences produced from the expression of a polynucleotide according to the present
disclosure, wherein ingestion of said ribonucleotide sequences by a Hemiptera crop
plant pest reduces feeding of said pest.
[0014] In a fourth aspect, embodiments of this disclosure provide vectors or expression
systems comprising a nucleic acid molecule according to the second aspect and to cells
transformed, transduced or transfected with said vector.
[0015] In a fifth aspect, some embodiments of this disclosure relate to plants transformed
with a polynucleotide according to the present disclosure, or a seed thereof comprising
said polynucleotide.
[0016] Further, some embodiments pertain to commodity products produced from a plant according
to the fifth aspect, wherein said commodity product comprises a detectable amount
of a polynucleotide according to the second aspect or a ribonucleotide expressed therefrom.
[0017] In a sixth aspect, some embodiments provide methods for controlling Hemiptera pest
infestation comprising providing in the diet of a Hemiptera pest an agent comprising
a first polynucleotide sequence that functions upon ingestion by the pest to inhibit
a biological function within said pest, wherein said polynucleotide sequence exhibits
from about 95 to about 100 percent nucleotide sequence identity along at least from
about 16 to about 25 contiguous nucleotides to a SHP coding sequence derived from
said pest and is hybridized to a second polynucleotide sequence that is complementary
to said first polynucleotide sequence, and wherein said coding sequence derived from
said pest comprise a sequence selected from the group consisting of SEQ ID NO:1, SEQ
ID NO:5 and SEQ ID NO:7 or a complement thereof.
[0018] Further, in a seventh aspect, embodiments of the present disclosure pertains to methods
for controlling a Hemiptera pest a plant cell expressing a polynucleotide sequence
according to the present disclosure, wherein the polynucleotide is expressed to produce
a double stranded ribonucleic acid, wherein said double stranded ribonucleotide acid
and/or a RNAi inducing compound derived from said double stranded ribonucleotide acid
functions upon ingestion by the pest to inhibit the expression of a SHP encoding target
sequence within said pest and results in decreased feeding on said diet relative to
a diet lacking the plant cell.
[0019] Further, in an eight aspect, embodiments of the present disclosure pertains to method
for improving the yield of a crop produced from a crop plant subjected to insect pest
infestation, said method comprising the steps of,
- a) introducing a polynucleotide according to the present disclosure into said crop
plant,
- b) cultivating the crop plant to allow the expression of said polynucleotide, wherein
expression of the polynucleotide inhibits feeding by insects pests and loss of yield
due to pest infestation.
[0020] In a further aspect, the present disclosure relates to transgenic plant comprising
a gene coding an inhibitor against SHP of a target pest.
[0021] Before the disclosure is described in detail, it is to be understood that this disclosure
is not limited to the particular component parts of the process steps of the methods
described. It is also to be understood that the terminology used herein is for purposes
of describing particular embodiments only, and is not intended to be limiting. It
must be noted that, as used in the specification and the appended claims, the singular
forms "a," "an" and "the" include singular and/or plural referents unless the context
clearly dictates otherwise. It is moreover to be understood that, in case parameter
ranges are given which are delimited by numeric values, the ranges are deemed to include
these limitation values.
BRIEF DESCRIPTION OF THE DRAWINGS
[0022]
Figure 1 represents electron microscopy pictures showing the Influence of SHP silencing
on sheath formation.
Figure 2 are two diagrams showing the temporal evolution of behavior (EPG wavepatterns)
SHP RNAi (B) aphids and (A) controls.
Figure 3 is a diagram showing percentage of important non-phloematic and phloematic
EPG wavepatterns.
Figure 4 is are two diagrams showing the reproduction SHP RNAi aphids and controls.
Figure 5 is a diagram showing the survival analysis of SHP RNAi aphids and controls
by Kaplan Meier Log-Rank.
Figure 6 is a nucleic acid sequence showing a part of the mRNA sequence coding for
the A.pisum SHP (SEQ ID NO.1).
Figure 7 is the nucleic acid sequence of a dsRNA (SEQ ID No. 2) derived from SEQ ID
No. 1 for A.pisum pest control.
DETAILED DESCRIPTION OF THE DISCLOSURE
[0023] Disclosed herein do novel pest control methods comprise the incorporation of an inhibitor
against the structural sheath protein (SHP) into the body of an agricultural target
pest, in particular against insect pests belonging to the order Hemiptera like aphids,
and to pest control agents to be used in the method and to transgenic crop, greenhouse
and ornamental plants.
[0024] Furthermore, the present disclosure provides methods and compositions for genetic
control of pest infestations. For example, the present disclosure provides recombinant
DNA technologies to post-transcriptionally repress or inhibit expression of a target
structural sheath protein (SHP) coding sequence in the cell of a pest to provide a
pest-protective effect by feeding to the pest one or more double stranded RNA (dsRNA)
and/or small interfering ribonucleic acid (siRNA) molecules transcribed from all or
a portion of a target coding sequence, thereby controlling the infestation. Therefore,
the present disclosure relates to sequence-specific inhibition of expression of SHP
coding sequences using double- stranded RNA (dsRNA), including small interfering RNA
(siRNA), to achieve the intended levels of pest control.
[0025] Isolated and substantially purified nucleic acid molecules including but not limited
to non-naturally occurring nucleotide sequences and recombinant DNA constructs for
transcribing dsRNA molecules of the present disclosure are provided that suppress
or inhibit the expression of target coding sequence for the structural sheath protein
(SHP) in the pest when introduced thereto. Transgenic plants that (a) contain nucleotide
sequences encoding the isolated and substantially purified nucleic acid molecules
and the non-naturally occurring recombinant DNA constructs for transcribing the dsRNA
molecules for controlling plant pest infestations, and (b) display resistance and/or
enhanced tolerance to the insect infestations, are also provided. Compositions containing
the dsRNA nucleotide sequences of the present disclosure for use in topical applications
onto plants or onto animals or into the environment of an animal to achieve the elimination
or reduction of pest infestation are also described.
[0026] Surprisingly, the inventors found that inhibiting SHP is a universally applicable
form of pest control. For example, the generation of transgenic plants expressing
dsRNA targeted at the SHP in specific insect pests is an efficient and environmentally
sustainable approach to reduce the impact of insect pests on agriculture.
[0027] SHP is responsible for hardening of the salivary sheath, a protein structure that
is formed out of gel saliva that is secreted during stylet movement through the plant
tissue (Tjallingii and Hogen Esch, 1993). The inventors approach was based on their
findings that salivary sheath were shown for a numerousness pest species, in particular
for a wide range of species belonging to the order Hemiptera like aphids. For example,
salivary sheath were shown for all aphid species studied so far and sequences with
a close similarity are present in M. persicae EST database (EST accessions EC387934,
EC388457 and EE572212 (http://www.ncbi.nlm.nih.gov/)). High sequence overlaps of mRNA
(RefSeq XM_001943863 (http://www.ncbi.nlm.nih.gov/)) of 99% were reported for the
species
Sitobion avenae and
Metopolophium dirhodum (Rao SAK (2011) "The identification and characterization of salivary proteins from
the cereal aphids Sitobion avenae, Metopolophium dirhodum and Rhopalosiphum padi",
PhD thesis, University College Dublin, Ireland).
[0028] Beside aphids, formation of a sheath-like structure could also be observed for other
groups of insects belonging to the order Hemiptera including Sternorrhyncha such as
whiteflies (Freeman et al., 2001) and for planthoppers (Fulgoromorpha) (Brentassi
et al., 2007). The two sister groups Sternorryhncha and Fulgoromorpha show an overlap
of protein sequence of the SHP protein that potentially originates from a common ancestor.
[0029] The inventor identified that that silencing of SHP in the insects, for example induced
by injection of specific double stranded RNA, prevents sheath hardening. This leads
to later and reduced feeding and a significantly reduced reproduction rate (-50%)
in comparison to control groups. It can be assumed that reduced feeding will decrease
negative influences on plant development due to reduced removal of nutrition by the
insects, in particular by aphids. In addition reduced feeding will lower the risk
of infection of aphid transmitted plant viruses like the barley yellow dwarf virus.
Reduced reproduction will also lead to a slow population growth that makes for example
single aphids easier to access to predators, e.g. ladybeetles.
[0030] The results according to the present disclosure indicate that a nucleotide sequence,
either DNA or RNA coding for SHP can be used to construct plant cells resistant to
infestation by the pest. The pest host, for example, can be transformed to contain
one or more of SHP encoding nucleotide sequences. The nucleotide sequence transformed
into the pest host or symbiont may encode one or more RNAs that form into a dsRNA
sequence in the cells or biological fluids within the transformed host or symbiont,
thus making the dsRNA available in the diet of the pest if/when the pest feeds upon
the transgenic host or symbiont, resulting in the suppression of expression of SHP
in the cells of the pest and ultimately the death, stunting, or other inhibition of
the pest.
[0031] Post-transcriptional gene silencing may be used to downregulate the expression of
the SHP coding gene. The gene silencing can be achieved e.g. by antisense molecules
or molecules that mediate RNA interference.
[0032] Antisense polynucleotides are designed to specifically bind to RNA, resulting in
the formation of RNA-DNA or RNA-RNA hybrids, with an arrest of reverse transcription
or messenger RNA translation. Many forms of antisense have been developed and can
be broadly categorized into enzyme-dependent antisense or steric blocking antisense.
Enzyme-dependent antisense includes forms dependent on RNase H activity to degrade
target mRNA, including single-stranded DNA, RNA, and phosphorothioate antisense. Antisense
polynucleotides are typically generated within the cell by expression from antisense
constructs that contain the antisense strand as the transcribed strand. Antisense
polynucleotides will bind and/or interfere with the translation of the corresponding
mRNA. Antisense RNA or antisense oligodeoxynucleotides (antisense ODNs) can both be
used and may also be prepared in vitro synthetically or by means of recombinant DNA
techniques. In order to avoid their digestion by DNAse, ODNs and antisense RNAs may
be chemically modified. Trans-cleaving catalytic RNAs (ribozymes) are RNA molecules
possessing endoribonuclease activity. Ribozymes are specifically designed for a particular
target, and the target message must contain a specific nucleotide sequence. They are
engineered to cleave any RNA species site-specifically in the background of cellular
RNA. The cleavage event renders the mRNA unstable and prevents protein expression.
[0033] In other advantageous embodiments the used methods for reducing SHP expression on
a post-transcriptional level are based on RNA interference (RNAi). Methods for downregulating
genes by RNAi are well known to the skilled person and thus, do not need any detailed
description here. Examples of RNAi inducing compounds that can be used to knockdown
the expression of the SHP encoding gene include but are not limited to short interfering
nucleic acids (siNA), short interfering RNA (siRNA), microRNA (miRNA), short hairpin
RNAs (shRNA) as well as precursors thereof which are processed in the cell to the
actual RNAi inducing compound. According to one embodiment, a siRNA is used for silencing.
The siRNA may be provided as double-stranded molecule having 3' overhangs on each
strand. Blunt ended molecules may also be used. Said siRNA may comprise desoxy- as
well as ribonucleotides and furthermore, may comprise modified nucleotides. Several
embodiments and variations of siRNA compounds are known in the prior art and can be
used to reduce expression of the SHP gene. In order to efficiently induce silencing,
the siRNA used as RNAi inducing compound is substantially complementary to a portion
of the target gene transcript for inhibiting the expression of said gene by RNA interference.
[0034] The present disclosure relates generally to genetic control of infestations in host
organisms belonging to the order Hemiptera. More particularly, the present disclosure
includes methods for delivery of pest control agents to an aphid pest. Such pest control
agents cause, directly or indirectly, an impairment in the ability of the pest to
maintain itself, grow or otherwise infest a target host or symbiont. The present disclosure
provides methods for employing stabilized dsRNA molecules in the diet of the pest
as a means for suppression of the targeted genes encoding SHP in the pest, thus achieving
desired control of pest infestations in, or about the host or symbiont targeted by
the pest.
[0035] In accomplishing the foregoing, the present disclosure provides methods of inhibiting
expression of the SHP encoding target gene in an insect pest, in particular in an
insect belonging to the order Hemiptera including insects belonging to the suborder
Sternorrhyncha and/or to the infraorder Fulgoromorpha, in particular in aphids like
Acyrthosiphon pisum, resulting in the cessation of feeding, growth, development, reproduction, infectivity,
and eventually may result in the death of the pest.
[0036] The method comprises in one embodiment introducing partial or fully stabilized double-stranded
RNA (dsRNA) nucleotide molecules into a nutritional composition that the pest relies
on as a food source, and making the nutritional composition available to the pest
for feeding. Ingestion of the nutritional composition containing the double stranded
or siRNA molecules results in the uptake of the molecules by the cells of the pest,
resulting in the inhibition of expression of at least one target gene in the cells
of the pest. Inhibition of the target gene exerts a deleterious effect upon the pest.
[0037] In certain embodiments, dsRNA molecules provided by the disclosure comprise nucleotide
sequences complementary to a nucleic acid sequence comprised in SEQ ID NO:1, the inhibition
of which in a pest organism results in the reduction or removal of SHP. The nucleotide
sequence selected may exhibit from about 80% to at least about 100% sequence identity
to 16 to 25 contiguous nucleotides of SEQ ID NO:1, including the complement thereof.
Such inhibition can be described as specific in that a nucleotide sequence from a
portion of the SHP encoding target gene is chosen from which the inhibitory dsRNA
or siRNA is transcribed. The method is effective in inhibiting the expression of the
SHP target gene and can be used to inhibit many different types of pests. In a particular
embodiment, the nucleotide sequence is SEQ ID NO:2.
[0038] In advantageous embodiments, the nucleic acid sequences identified as having a pest
protective effect may be readily expressed as dsRNA molecules through the creation
of appropriate expression constructs. For example, such sequences can be expressed
as a hairpin and stem and loop structure by taking a first segment corresponding to
SEQ ID NO:1 or a fragment thereof, linking this sequence to a second segment spacer
region that is not homologous or complementary to the first segment, and linking this
to a third segment that transcribes an RNA, wherein at least a portion of the third
segment is substantially complementary to the first segment. Such a construct forms
a stem and loop structure by hybridization of the first segment with the third segment
and a loop structure forms comprising the second segment (
WO94/01550 WO98/05770,
US 2002/0048814AI, and
US 2003/0018993 Al).
A. Definitions
[0039] As used in the present disclosure, "cell", "cell line", and "cell culture" can be
used interchangeably and all such designations include progeny. Thus, the words "transformants"
or "transformed cells" includes the primary subject cell and cultures derived therefrom
without regard for the number of transfers. It is also understood that all progeny
may not be precisely identical in DNA content, due to deliberate or inadvertent mutations.
Mutant progeny that have the same functionality as screened for in the originally
transformed cell are included.
[0040] As used herein, the phrase "coding sequence", "encoding sequence", "structural nucleotide
sequence" or "structural nucleic acid molecule" refers to a nucleotide sequence that
is translated into a polypeptide, usually via mRNA, when placed under the control
of appropriate regulatory sequences. The boundaries of the coding sequence are determined
by a translation start codon at the 5'-termmus and a translation stop codon at the
3'-terminus. A coding sequence can include, but is not limited to, genomic DNA5 cDNA,
EST and recombinant nucleotide sequences.
[0041] The term "complementary" as used herein refers to a relationship between two nucleic
acid sequences. One nucleic acid sequence is complementary to a second nucleic acid
sequence if it is capable of forming a duplex with the second nucleic acid, wherein
each residue of the duplex forms a guanosine-cytidine (G-C) or adenosine-thymidine
(A-T) base pair or an equivalent base pair. Equivalent base pairs can include nucleoside
or nucleotide analogues other than guanosine, cytidine, adenosine, or thymidine.
[0042] The term "derivative" as used herein, refers to a nucleic acid molecule that has
similar binding characteristics to the SHP target nucleic acid sequence as a nucleic
acid molecule according to one of the claimed sequences.
[0043] As used herein, the term "derived from" refers to a specified nucleotide sequence
that may be obtained from a particular specified source or species, albeit not necessarily
directly from that specified source or species.
[0044] The term "expression clone" refers to DNA sequences containing a desired coding sequence
and control sequences in operable linkage, so that hosts transformed with these sequences
are capable of producing the encoded proteins. The term "expression system" refers
to a host transformed with an expression clone. To effect transformation, the expression
clone may be included on a vector; however, the relevant DNA may also be integrated
into the host chromosome.
[0045] The term "gene" refers to a DNA sequence that comprises control and coding sequences
necessary for the production of a recoverable bioactive polypeptide or precursor.
[0046] The term "homologue of the nucleic acid molecule" refers to a nucleic acid molecule
the sequence of which has one or more nucleotides added, deleted, substituted or otherwise
chemically modified in comparison to a nucleic acid molecule according to one of the
claimed sequences, provided always that the homologue retains substantially the same
inhibitory effect on SHP.
[0047] The term "isolated" describes any molecule separated from its natural source.
[0048] As used herein, the term "nucleic acid" refers to a single or double-stranded polymer
of deoxyribonucleotide or ribonucleotide bases read from the 5' to the 3' end. The
"nucleic acid" may also optionally contain non-naturally occurring or altered nucleotide
bases that permit correct read through by a polymerase and do not reduce expression
of a polypeptide encoded by that nucleic acid. The term "nucleotide sequence" or "nucleic
acid sequence" refers to both the sense and antisense strands of a nucleic acid as
either individual single strands or in the duplex. The term "ribonucleic acid" (RNA)
is inclusive of RNAi (inhibitory RNA), dsRNA (double stranded RNA), siRNA (small interfering
RNA), mRNA (messenger RNA), miRNA (micro-RNA), tRNA (transfer RNA, whether charged
or discharged with a corresponding acylated amino acid), and cRNA (complementary RNA)
and the term "deoxyribonucleic acid" (DNA) is inclusive of cDNA and genomic DNA and
DNA-RNA hybrids. The words "nucleic acid segment", "nucleotide sequence segment",
or more generally "segment" will be understood by those in the art as a functional
term that includes both genomic sequences, ribosomal RNA sequences, transfer RNA sequences,
messenger RNA sequences, operon sequences and smaller engineered nucleotide sequences
that express or may be adapted to express, proteins, polypeptides or peptides.
[0049] Provided according to the disclosure are nucleotide sequences, the expression of
which results in an RNA sequence which is substantially homologous to an RNA molecule
of a targeted gene encoding SHP in an insect that comprises an RNA sequence encoded
by a nucleotide sequence within the genome of the insect. Thus, after ingestion of
the stabilized RNA sequence down- regulation of the nucleotide sequence of the target
gene in the cells of the insect may be obtained resulting in a deleterious effect
on the maintenance, viability, proliferation, reproduction and infestation of the
insect.
[0050] As used herein, the term "homologous" or "homologs", with reference to a nucleic
acid sequence, includes a nucleotide sequence that hybridizes under stringent conditions
to one of the coding sequences of SEQ ID NO:1, SEQ ID NO. 5 or SEQ ID NO. 7, or the
complements thereof. Sequences that hybridize for example under stringent conditions
to SEQ ID NO:1, or the complements thereof, are those that allow an antiparallel alignment
to take place between the two sequences, and the two sequences are then able, under
stringent conditions, to form hydrogen bonds with corresponding bases on the opposite
strand to form a duplex molecule that is sufficiently stable under the stringent conditions
to be detectable using methods well known in the art. Substantially homologous sequences
have preferably from about 70% to about 80% sequence identity, or more preferably
from about 80% to about 85% sequence identity, or most preferable from about 90% to
about 95% sequence identity, to about 99% sequence identity, to the referent nucleotide
sequences of SEQ ID NO:1, SEQ ID NO. 5 or SEQ ID NO. 7, or to the sequence of SEQ
ID NO:2 as set forth in the sequence listing, or the complements thereof.
[0051] As used herein, the term "insect control agent", or "gene suppression agent" refers
to a particular RNA molecule comprising a first RNA segment and a second RNA segment,
wherein the complementarity between the first and the second RNA segments results
in the ability of the two segments to hybridize in vivo and in vitro to form a double
stranded molecule. It may generally be preferable to include a third RNA segment linking
and stabilizing the first and second sequences such that the entire structure forms
into a stem and loop structure, or even more tightly hybridizing structures may form
into a stem-loop knotted structure. Alternatively, a symmetrical hairpin could be
formed without a third segment in which there is no designed loop, but for steric
reasons a hairpin would create its own loop when the stem is long enough to stabilize
itself. The first and the second RNA segments will generally be within the length
of the RNA molecule and are substantially inverted repeats of each other and linked
together by the third RNA segment. The first and the second segments correspond invariably
and not respectively to a sense and an antisense sequence with respect to the target
RNA transcribed fern the target gene in the target insect pest that is suppressed
by the ingestion of the dsRNA molecule. The insect control agent can also be a substantially
purified (or isolated) nucleic acid molecule and more specifically nucleic acid molecules
or nucleic acid fragment molecules thereof from a genomic DNA (gDNA) or cDNA library.
Alternatively, the fragments may comprise smaller oligonucleotides having from about
15 to about 250 nucleotide residues, and more preferably, about 15 to about 30 nucleotide
residues.
[0052] As used herein, the phrase "inhibition of gene expression" or "inhibiting expression
of a target gene in the cell of an insect" refers to the absence (or observable decrease)
in the level of protein and/or mRNA product from the target gene. Specificity refers
to the ability to inhibit the target gene without manifest effects on other genes
of the cell and without any effects on any gene within the cell that is producing
the dsRNA molecule. The inhibition of gene expression of the target gene in the insect
pest may result in novel phenotypic traits in the insect pest.
[0053] The term "microorganism" includes prokaryotic and eukaryotic microbial species such
as bacteria, fungi and algae. Fungi include yeasts and filamentous fungi, among others.
Illustrative prokaryotes, both Gram-negative and Gram-positive, include Enter obacteriaceae,
such as Escherichia, Erwinia, Shigella, Salmonella, and Proteus; Bacillaceae; Rhizobiceae,
such as Rhizobium; Spirillaceae, such as photobacterium, Zymomonas, Serratia, Aeromonas,
Vibrio, Desulfovibrio, Spirillum', Lactobacillaceae; Pseudomoriadaceae, such as Pseudomonas
and Acetobacter; Azotobacteraceae, Actinomycetales, and Nitrobacteraceae. Among eukaryotes
are fungi, such as Phycomycetes and Ascomycetes, which includes yeast, such as Saccharomyces
and Schizosaccharomyces; and Basidiomycetes, such as Rhodotorula, Aureobasidium, Sporobolomyces,
and the like.
[0054] The term "operably linked", as used in reference to a regulatory sequence and a structural
nucleotide sequence, means that the regulatory sequence causes regulated expression
of the linked structural nucleotide sequence. "Regulatory sequences" or "control elements"
refer to nucleotide sequences located upstream (5' noncoding sequences), within, or
downstream (3' non-translated sequences) of a structural nucleotide sequence, and
which influence the timing and level or amount of transcription, RNA processing or
stability, or translation of the associated structural nucleotide sequence. Regulatory
sequences may include promoters, translation leader sequences, introns, enhancers,
stem-loop structures, repressor binding sequences, and polyadenylation recognition
sequences and the like.
[0055] In the present description, "pest control" refers to the removal or the reduction
of harm of pests. The concept of "pest control" include reducing feeding of the target
pest, killing of pests (extermination), pest proliferation inhibition, pest growth
inhibition, repelling of pests (repellence), and the removal or the reduction of harm
of pests (for example, inhibition of ingestion capacity of agricultural pests.
[0056] The term "plant" includes the plant body, plant organs (for example, leaves, petals,
stem, root, rhizome, and seeds), plant tissues (for example, epidermis, phloem, parenchyma,
xylem, and vascular bundle), and plant cells. In addition, the term "plant cell" includes
seed suspension cultures, embryos, meristematic tissue regions, callus tissues, cells
derived from leaves and roots, and gametophytes (embryos and pollens) and their precursors.
When plant culture cells are transformed, an organ or individual is regenerated from
the transformed cells by a known tissue culture method. These operations are readily
performed by those skilled in the art. An example is described below. Firstly, the
transformed plant cells are cultured in a sterilized callus forming medium (containing
a carbon source, saccharides, vitamins, inorganics, and phytohormones such as auxin
and cytokinin), thereby forming a dedifferentiated calluse which indefinitely proliferates
(callus induction). The formed callus is transferred to a new medium containing a
plant growth regulator such as auxin, and further proliferated thereon (subcultivation).
When the callus induction is carried out on a solid medium such as agar and subcultivation
is carried out in a liquid medium, the respective cultures are efficiently achieved.
Secondly, the callus proliferated by subcultivation was cultured under appropriate
conditions, thereby inducing redifferentiation of the organ (inductive redifferentiation),
and regenerating the plant body. The inductive redifferentiation is achieved by appropriately
adjusting the type and amount of the various components of the medium, including plant
growth regulators such as auxin and cytokinin, and the carbon source, and the light
and temperature. The inductive redifferentiation forms adventitious embryos, adventitious
roots, adventitious buds, adventitious foliage, and others, and they are grown into
a complete plant body. The plant before being a complete plant body may be stored
in the form of, for example, capsulated artificial seeds, dry embryos, lyophilized
cells, or tissues.
[0057] The term "plasmid", "vector system", "vector" or "expression vector" means a construct
capable of
in vivo or
in vitro expression. In the context of the present disclosure, these constructs may be used
to introduce genes encoding enzymes into host cells.
[0058] The term "polynucleotide" corresponds to any genetic material of any length and any
sequence, comprising single-stranded and double-stranded DNA and RNA molecules, including
regulatory elements, structural genes, groups of genes, plasmids, whole genomes and
fragments thereof.
[0059] The term "recombinant DNA" or "recombinant nucleotide sequence" refers to DNA that
contains a genetically engineered modification through manipulation via mutagenesis,
restriction enzymes, and the like.
[0060] The term "stringent conditions" relates to conditions under which a probe will hybridize
to its target subsequence, but to no other sequences. Stringent conditions are sequence-dependent
and will be different in different circumstances. Longer sequences hybridize specifically
at higher temperatures. Generally, stringent conditions are selected to be about 5°
C lower than the thermal melting point (Tm) for the specific sequence at a defined
ionic strength and pH. The Tm is the temperature (under defined ionic strength, pH
and nucleic acid concentration) at which 50% of the probes complementary to the target
sequence hybridize to the target sequence at equilibrium. (As the target sequences
are generally present in excess, at Tm, 50% of the probes are occupied at equilibrium).
Typically, stringent conditions will be those in which the salt concentration is less
than about 1.0 M Na ion, typically about 0.01 to 1.0 M Na ion (or other salts) at
pH 7.0 to 8.3 and the temperature is at least about 30° C for short probes (e.g. 10
to 50 nucleotides) and at least about 60° C for longer probes. Stringent conditions
may also be achieved with the addition of destabilizing agents, such as formamide
and the like.
[0061] As used herein, the term "sequence identity", "sequence similarity" or "homology"
is used to describe sequence relationships between two or more nucleotide sequences.
The percentage of "sequence identity" between two sequences is determined by comparing
two optimally aligned sequences over a comparison window, wherein the portion of the
sequence in the comparison window may comprise additions or deletions (i.e., gaps)
as compared to the reference sequence (which does not comprise additions or deletions)
for optimal alignment of the two sequences. The percentage is calculated by determining
the number of positions at which the identical nucleic acid base or amino acid residue
occurs in both sequences to yield the number of matched positions, dividing the number
of matched positions by the total number of positions in the window of comparison,
and multiplying the result by 100 to yield the percentage of sequence identity. A
sequence that is identical at every position in comparison to a reference sequence
is said to be identical to the reference sequence and vice-versa. A first nucleotide
sequence when observed in the 5' to 3' direction is said to be a "complement" of,
or complementary to, a second or reference nucleotide sequence observed in the 3'
to 5' direction if the first nucleotide sequence exhibits complete complementarity
with the second or reference sequence. As used herein, nucleic acid sequence molecules
are said to exhibit "complete complementarity" when every nucleotide of one of the
sequences read 5' to 3' is complementary to every nucleotide of the other sequence
when read 3' to 5'. A nucleotide sequence that is complementary to a reference nucleotide
sequence will exhibit a sequence identical to the reverse complement sequence of the
reference nucleotide sequence. These terms and descriptions are well defined in the
art and are easily understood by those of ordinary skill in the art.
[0062] By "synergistic" it is meant to include the synergistic effects of the combination
on the pesticidal activity (or efficacy) of the combination of the transgenic event
and the pesticide. However, it is not intended that such synergistic effects be limited
to the pesticidal activity, but that they should also include such unexpected advantages
as increased scope of activity, advantageous activity profile as related to type and
amount of damage reduction, decreased cost of pesticide and application, decreased
pesticide distribution in the environment, decreased pesticide exposure of personnel
who produce, handle and plant corn seeds, and other advantages known to those skilled
in the art.
[0063] The term "variant of the nucleic acid molecule" refers herein to a nucleic acid molecule
which is substantially similar in structure and biological activity to a nucleic acid
molecule according to one of the claimed sequences.
[0064] The "pest" refers to the pest subjected to pest control, or the pest controlled by
the present disclosure. The pest may be two or more pests and are not particularly
limited. In general, pests are broadly divided into agricultural pests, sanitary pests,
and unpleasant pests. "Agricultural pests" refer to the pests that attack crops (including
garden crops and crops during storage). "Sanitary pests" refer to the pests that attack
the sanitary environment of human. In addition, "unpleasant pests" refer to the pests
that attack the mood of human by their appearance or motion. The present disclosure
is also applicable to the pests that attack the assets of human (for example, termite
and bristletail) and livestock (for example, mosquito and parasite).
[0065] Therefore, as used herein, the term "target pest" refers to insects, arachnids, crustaceans,
fungi, bacteria, viruses, nematodes, flatworms, roundworms, pinworms, hookworms, tapeworms,
trypanosomes, schistosomes, botflies, fleas, ticks, mites, and lice and the like that
are pervasive in the human environment and that may ingest or contact one or more
cells, tissues, or fluids produced by a pest host or symbiont transformed to express
or coated with a double stranded gene suppression agent or that may ingest plant material
containing the gene suppression agent.
[0066] As used herein, a "pest resistance" trait is a characteristic of a transgenic plant,
transgenic animal, transgenic host or transgenic symbiont that causes the plant, animal,
host, or symbiont to be resistant to attack from a pest that typically is capable
of inflicting damage or loss to the plant, animal, host or symbiont. Such pest resistance
can arise from a natural mutation or more typically from incorporation of recombinant
DNA that confers pest resistance. To impart insect resistance to a transgenic plant
a recombinant DNA can, for example, be transcribed into a RNA molecule that forms
a dsRNA molecule within the tissues or fluids of the recombinant plant. The dsRNA
molecule is comprised in part of a segment of RNA that is identical to a corresponding
RNA segment encoded from a DNA sequence within an insect pest that prefers to feed
on the recombinant plant. Expression of the gene within the target insect pest is
suppressed by the dsRNA, and the suppression of expression of the gene in the target
insect pest results in the plant being insect resistant. Fire et al. (
U.S. Patent No. 6,506,599) generically described inhibition of pest infestation, providing specifics only about
several nucleotide sequences that were effective for inhibition of gene function in
the nematode species Caenorhabditis elegans. Similarly, Plaetinck et al. (
US 2003/0061626) describe the use of dsRNA for inhibiting gene function in a variety of nematode
pests. Mesa et al. (
US 2003/0150017) describe using dsDNA sequences to transform host cells to express corresponding
dsRNA sequences that are substantially identical to target sequences in specific pathogens,
and particularly describe constructing recombinant plants expressing such dsRNA sequences
for ingestion by various plant pests, facilitating down-regulation of a gene in the
genome of the pest and improving the resistance of the plant to the pest infestation.
B. Target Pests
[0067] The present disclosure pertains to pest control methods comprising incorporating
an inhibitor against the structural sheath protein (SHP) into the body of an agricultural
target pest expressing SHP. In particular, the mRNA encoding the SHP comprises the
sequence set forth in SEQ ID NO: 1, or homologs thereof, wherein said homologs may
have a sequence identity of at least 80 %, in particular of at least 85%, in particular
of at least 90% to SEQ ID NO: 1. Examples for such homologs are shown in Rao SAK (2011).
In an advantageous example said homologs are parts of sequences that encode a functional
SHP in the target pest.
[0068] In advantageous embodiments of the present disclosure, the target pests are insect
belonging to the insect order Hemiptera also known as the true bugs. Many Hemipteran
insects are important agricultural pests because they cause direct feeding damage
to their host plants and/or transmit plant disease agents including viruses and bacteria.
Microscopic and behavioral studies on different Hemiptera species showed that their
exuviae (molted skins) normally had either fully or partially extended stylets in
a feeding-like position. In most cases these stylets were still partially embedded
in their host plants after ecdysis, which indicated that plant-feeding hemipteran
nymphs use their stylets to anchor themselves to host plants during molting.
[0069] As an example, aphids feed by sucking the sugary sap from the phloem sieve tubes
of higher plants through specially adapted mouthparts known as stylets. Before feeding
can take place, the stylet must penetrate the plant epidermis and propagate through
the cortical layer. To facilitate this process, aphids secrete gel saliva that hardens
to form a surface flange and a continuous tubular sheath encasing the full length
of the stylet in the apoplast. Traces of gel saliva form in artificial diet a structure
reminiscent of a pearl necklace, indicating that the salivary sheath is formed progressively
from drops of saliva that harden rapidly. During stylet propagation, the continuous
sheath around the stylet provides mechanical stability and protect against chemical
defenses. The structural sheath protein (SHP) is responsible for hardening of the
salivary sheath.
[0070] Other hemiptera including plant-sucking insects such as whiteflies and planthoppers
also form with SHP a salivary sheath by secretion of gel saliva (Freeman et al., 2001
and Brentassi et al., 2007) and show feeding associated secretion of watery saliva.
[0071] Examples of the target pests belonging to Hemiptera include insects of the suborder
Sternorrhyncha including aphids and the infraorder Fulgoromorpha. In particular, examples
of the target pests belonging to Hemiptera include
Nilaparvata lugens, Sogatella furcifera, Laodelphax stratella, Nephotettix cincticeps,
Recilia dorsalis, Stenotus rubrovittatus, Trigonotylus caelestialium, Leptocorisa
chinensis, Nezara antennata, Nezara viridula, Lagynotomus elongatus, Scotinophara
lurida, Eysarcoris annamita, Eysarcoris lewisi, Eysarcoris ventralis, Togo hemipterus
Scott, Cletus punctiger, Piezodorus hybneri, Halyomorpha halys, Dolycoris baccarum,
Neotoxoptera formosana, Rhopalosiphum padi, Rhopalosiphum maidis, Acyrthosiphon pisum and
Aphis glycines.
[0072] In advantageous embodiments, the target pests are belonging to the genera of aphids,
in particular
Acyrthosiphon pisum.
C. SHP Inhibitor
[0073] According to the pest control methods of the present disclosure, an inhibitor against
SHP is incorporated into the body of the target pest. The term "SHP inhibitor" is
used as the generic name of the substances inhibiting SHP. The SHP inhibitor may be
of any type as long as it has inhibitory against the expression, the transcription
and/or the translation of SHP and/or has inhibitory activity against SHP.
[0074] Examples of the SHP inhibitor include a nucleic acid that inhibits the expression
of the SHP gene, and a substance that specifically binds to SHP (for example, an antibody
or a low molecular weight compound). The former one is further described below. The
substance that specifically binds to SHP may be obtained or prepared using binding
assay targeted at SHP. An antibody that specifically binds to SHP may be prepared
using, for example, an immunological method, a phage display method, or a ribosome
display method.
[0075] According to one aspect of the present disclosure, a compound selected from the group
consisting of the following (a) to (d) is used as the SHP inhibitor:
- (a) a RNAi inducing compound targeted a nucleic acid coding SHP or parts thereof;
- (b) a nucleic acid construct intracellularly producing a RNAi inducing compound targeted
a nucleic acid coding SHP or parts thereof;
- (c) an antisense nucleic acid targeted at the transcript product of a gene coding
SHP of the target pest; and
- (d) a ribozyme targeted at the transcript product of a gene coding SHP of the target
pest.
[0076] The (a) and (b) are the compounds used for the inhibition of expression by so-called
RNAi (RNA interference). In other words, when the compound (a) or (b) is used, the
expression of SHP is inhibited by RNAi, whereby pest control effect is achieved. In
this manner, the use of RNAi allows specific control of the target pest, and facilitates
rapid achievement of pest control effect. Furthermore, owing to its properties, the
possibility of occurrence of resistant strains is likely extremely low. In addition,
RNAi does not modify plant genes, and thus will not genetically influence them.
[0077] The "RNAi" refers to the inhibition of expression of the target gene by the introduction
of an RNA composed of a sequence homologous to that of the target gene (specifically
homologue to the mRNA corresponding to the target gene) into the target cell. For
the inhibition of expression using the RNAi method in pests such as insects, generally,
a dsRNA (double strand RNA) composed of a sequence corresponding a part of the target
gene (the gene coding the IAP of the target pest). Two or more dsRNAs may be used
for one target gene.
[0078] The RNAi targeted at the gene of a mammal cell uses a short dsRNA (siRNA) of about
16 to 25 nucleotides. When the RNAi is targeted at the gene of a pest such as an insect,
a long dsRNA of more than several hundreds of nucleotides is preferred because owing
to its effectiveness. The length of the dsRNA used for RNAi is, for example, 30 nucleotides
or more, and preferably 200 nucleotides or more (for example, from 200 to 500 nucleotides).
The use of a dsRNA is preferred for inducing effective inhibition of expression, but
the use of a single strand RNA will also be contemplated. The dsRNA used herein is
not necessarily composed of two molecules of sense and antisense strands, and, for
example, may have a structure wherein the sense and antisense strands composing the
dsRNA are connected via a hairpin loop. A dsRNA composed of a modified RNA may be
used. Examples of the modification include phosphorothioation, and the use of a modified
base (for example, fluorescence-labeled base). In advantageous embodiments, the RNAi
inducing compound is a compound selected from the group consisting of short interfering
nucleic acids, siNA, short interfering RNA (siRNA), microRNA (miRNA), short hairpin
RNAs (shRNA) and precursors thereof which are processed in the cell to the actual
RNAi inducing compound. In a preferred embodiment, the precursor is double-stranded
RNA (dsRNA). An example of a dsRNA used in the pest control method according to the
present disclosure is a dsRNA comprising the sequence set forth in SEQ ID NO: 2, or
homologs thereof, wherein said homologs have a sequence identity of at least 80 %,
in particular of at least 85%, in particular of at least 90% to SEQ ID NO: 2.
[0079] An RNAi specific to the target gene can be also produced by intracellularly expression
of a dsRNA targeted at the target gene. The nucleic acid construct (b) is used as
such a means.
[0080] The dsRNA used in the RNAi method may be prepared by chemical synthesis, or
in vitro or
in vivo using an appropriate expression vector. The method using an expression vector is
particularly effective for the preparation of a relatively long dsRNA. The design
of dsRNA normally includes the sequence (continuous sequence) specific to the target
nucleic acid. Programs and algorithms for selecting an appropriate target sequence
have been developed.
[0081] As mentioned above, advantageous embodiments of the present disclosure pertain to
the use of RNA interference to silence the expression of SHP to disrupt the sheath
formation and therefore the insect feeding and reproduction were inhibited.
[0082] The above described (c) is a compound used for the inhibition of expression by an
antisense method. The inhibition of expression using an antisense method is generally
carried out using an antisense construct that produces a RNA complementary to the
portion specific to the mRNA corresponding to the target gene upon transcription.
The antisense construct (also referred to as antisense nucleic acid) is, for example,
introduced into the target cell in the form of an expression plasmid. The antisense
construct may be an oligonucleotide probe that hybridizes with the DNA sequence or
corresponding mRNA sequence of the target gene (these sequences may be collectively
referred to as "target nucleic acid") upon introduction into the target cell, and
inhibits their expression. The oligonucleotide probe is preferably resistant to endogenous
nucleases such as exonuclease and/or endonuclease. When a DNA molecule is used as
an antisense nucleic acid, the DNA molecule is preferably an oligodeoxyribonucleotide
derived from the region containing the translation initiation site of the mRNA corresponding
to the target gene (for example, the region from -10 to +10).
[0083] The complementation between the antisense nucleic acid and target nucleic acid is
preferably precise, but some mismatch may occur. The hybridization capacity of the
antisense nucleic acid for the target nucleic acid generally depends on the degree
of complementation between the nucleic acids and the length of the antisense nucleic
acid. In principle, the longer the antisense nucleic acid, the more stable double
strand (or triplex) is formed between the antisense and target nucleic acids, even
if many mismatches occur. Those skilled in the art can examine the degree of acceptable
mismatch using a standard method.
[0084] The antisense nucleic acid may be DNA, RNA, or a chimera mixture thereof, or a derivative
or modified product thereof. The antisense nucleic acid may be single or double strand.
The stability and hybridization capacity of the antisense nucleic acid are improved
by the modification of the base, sugar, or phosphoric acid backbone. The antisense
nucleic acid may be synthesized by an ordinary method using, for example, a commercially
available automatic DNA synthesizing apparatus (for example, manufactured by Applied
Biosystems). The preparation of the modified nucleic acid and derivatives may refer
to, for example, Stein et al. (1988), Nucl. Acids Res. 16:3209 or Sarin et al., (1988),
Proc. Natl. Acad. Sci. U.S.A. 85:7448-7451.
[0085] In order to improve the action of the antisense nucleic acid in the target cell,
a promoter (for example, actin promoter or ie1 promoter) that strongly acts in the
target cell may be used. More specifically, when a construct containing the antisense
nucleic acid under control of the promoter is introduced into the target cell, a sufficient
amount of antisense nucleic acid is transcribed.
[0086] According to one aspect of the present disclosure, the inhibition of expression by
ribozyme is used (when the compound (d) is used). The mRNA corresponding to the target
gene may be destroyed using a ribozyme that cleaves the mRNA at the site-specific
recognition sequence, but preferably a hammerhead ribozyme is used. The method for
constructing the hammerhead ribozyme may be referred to, for example, Haseloff and
Gerlach, 1988,
Nature, 334:585-591.
[0087] In the same manner as in the antisense method, for example, for the purpose of improving
stability and target performance, the ribozyme construction may use a modified oligonucleotide.
In order to produce an effective amount of ribozyme within the target cell, it is
preferred that a nucleic acid construct including DNA coding the ribozyme be used
under the control of a promoter which strongly acts in insect cells (for example,
an actin promoter or an ie1 promoter).
SEQ ID NO. 1 shows a part of a mRNA sequence (NA 647-4776) encoding SHP in Acyrthosiphon pisum (SEQ ID NOs: 1).
SEQ ID NO: 2 is a dsRNA derived from SEQ ID NO.1.
SEQ ID NO: 3 is a plasmid nucleic acid sequence after cloning a dsRNA production vector.
SEQ ID NO: 4 is an amino acid sequence comprised in SHP from Acyrthosiphon pisum.
SEQ ID NO: 5 is a nucleic acid sequence comprised in a SHP mRNA from Sitobion avenae and SEQ ID NO: 6 is the corresponding amino acid sequence comprised in the encoded
protein.
SEQ ID NO: 7 is a nucleic acid sequence comprised in a SHP mRNA from Metopolophium dirhodum and SEQ ID NO: 8 is the corresponding amino acid sequence comprised in the encoded
protein.
D. Nucleic Acid Compositions and Constructs
[0088] The present disclosure provides recombinant DNA constructs for use in achieving stable
transformation of particular host or symbiont pest targets. Transformed host or symbiont
pest targets may express pesticidally effective levels of preferred dsRNA or siRNA
molecules from the recombinant DNA constructs, and provide the molecules in the diet
of the pest. Pairs of isolated and purified nucleotide sequences may be provided from
cDNA library and/or genomic library information. The pairs of nucleotide sequences
may be derived from any preferred coleopteran pest for use as thermal amplification
primers to generate DNA templates for the preparation of dsRNA and siRNA molecules
of the present disclosure.
[0089] Provided according to the present disclosure are nucleotide sequences, the expression
of which results in an RNA sequence which is substantially homologous to an RNA molecule
of a targeted gene in an insect that comprises an RNA sequence encoded by a nucleotide
sequence within the genome of the insect. Thus, after ingestion of the stabilized
RNA sequence down- regulation of the nucleotide sequence of the target gene in the
cells of the insect may be obtained resulting in a deleterious effect on the maintenance,
viability, proliferation, reproduction and infestation of the insect.
[0090] Examples of isolated polynucleotide suitable as a pest control agent against a target
pest are the following (A) to (d):
- a) a polynucleotide comprising a nucleic acid sequence of SEQ ID NO:1;
- b) a polynucleotide that hybridizes to a nucleic acid sequence of SEQ ID NO:1 under
stringent conditions;
- c) a polynucleotide of at least 70, at least 80, at least 85, at least 90 percent
sequence identity, to a nucleic acid sequence of SEQ ID NO:1;
- d) a fragment of at least 16 contiguous nucleotides of a nucleic acid sequence of
SEQ ID NO:1; and
- e) a complement of the sequence of (a), (b), (c) or (d),
wherein ingestion by a Hemiptera crop plant pest of a double stranded ribonucleotide
sequence comprising at least one strand that is complementary to said polynucleotide
or said fragment reduce feeding of said pest.
[0091] Further provided by the disclosure is a fragment or concatemer of a nucleic acid
sequence of SEQ ID NO:1. The fragment may be defined as causing the death, inhibition,
stunting, or cessation of feeding of a pest when expressed as a dsRNA and provided
to the pest. The fragment may, for example, comprise at least about 16,17,18 19, 21,
23, 25, 40, 60, 80, 100, 125 or more contiguous nucleotides of the sequence set force
in SEQ ID NO:1, or a complement thereof. One beneficial DNA segment for use in the
present disclosure is at least from about 19 to about 23, or about 23 to about 100
nucleotides up to about 2000 nucleotides or more in length. Particularly useful will
be dsRNA sequences including about 23 to about 300 nucleotides homologous to a pest
target sequence. The disclosure also provides a ribonucleic acid expressed from any
of such sequences including a dsRNA. A sequence selected for use in expression of
a gene suppression agent can be constructed from a single sequence derived from one
or more target pests and intended for use in expression of an RNA that functions in
the suppression of a single gene or gene family in the one or more target pests, or
that the DNA sequence can be constructed as a chimera from a plurality of DNA sequences.
[0092] In further embodiments, the disclosure pertains to recombinant DNA constructs comprising
a nucleic acid molecule encoding a dsRNA molecule described herein. The dsRNA may
be formed by transcription of one strand of the dsRNA molecule from a nucleotide sequence
which is at least from about 80% to about 100% identical to a nucleotide sequence
comprising SEQ ID NO:1. Such recombinant DNA constructs may be defined as producing
dsRNA molecules capable of inhibiting the expression of endogenous target gene(s)
in a pest cell upon ingestion. The construct may comprise a nucleotide sequence of
the plant operably linked to a promoter sequence that functions in the host cell.
Such a promoter may be tissue-specific and may, for example, be specific to a tissue
type which is the subject of pest attack. In the case of rootworms, for example, it
may be desired to use a promoter providing root-preferred expression.
[0093] Nucleic acid constructs in accordance with the disclosure may comprise at least one
non-naturally occurring nucleotide sequence that can be transcribed into a single
stranded RNA capable of forming a dsRNA molecule in vivo through hybridization. Such
dsRNA sequences self-assemble and can be provided in the diet of a coleopteran pest
to achieve the desired inhibition.
[0094] A recombinant DNA construct may comprise two different non-naturally occurring sequences
which, when expressed in vivo as dsRNA sequences and provided in the diet of a coleopteran
pest, inhibit the expression of at least two different target genes in the cell of
the coleopteran pest. In certain embodiments, at least 3, 4, 5, 6, 8 or 10 or more
different dsRNAs are produced in a cell or plant comprising the cell that has a pest-inhibitory
effect. The dsRNAs may expressed from multiple constructs introduced in different
transformation events or could be introduced on a single nucleic acid molecule. The
dsRNAs may be expressed using a single promoter or multiple promoters. Li one embodiments
of the disclosure, single dsRNAs are produced that comprise nucleic acids homologous
to multiple loci within a pest. hi still yet another aspect, the disclosure provides
a recombinant host cell having in its genome at least one recombinant DNA sequence
that is transcribed to produce at least one dsRNA molecule that functions when ingested
by a coleopteran pest to inhibit the expression of a target gene in the pest. The
dsRNA molecule may be encoded by any of the nucleic acids described herein and as
set forth in the sequence listing. The present disclosure also provides a transformed
plant cell having in its genome at least one recombinant DNA sequence described herein.
Transgenic plants comprising such a transformed plant cell are also provided, including
progeny plants of any generation, seeds, and plant products, each comprising the recombinant
DNA.
[0095] The present disclosure provides DNA sequences capable of being expressed as a RNA
in a cell or microorganism to inhibit target gene expression in a cell, tissue or
organ of an insect. The sequences comprises a DNA molecule coding for one or more
different nucleotide sequences, wherein each of the different nucleotide sequences
comprises a sense nucleotide sequence and an antisense nucleotide sequence connected
by a spacer sequence coding for a dsRNA molecule of the present disclosure. The spacer
sequence constitutes part of the sense nucleotide sequence or the antisense nucleotide
sequence and forms within the dsRNA molecule between the sense and antisense sequences.
The sense nucleotide sequence or the antisense nucleotide sequence is substantially
identical to the nucleotide sequence of the target gene or a derivative thereof or
a complementary sequence thereto. The dsDNA molecule may be placed operably under
the control of a promoter sequence that functions in the cell, tissue or organ of
the host expressing the dsDNA to produce dsRNA molecules. In one embodiment, the DNA
sequence may be derived from a nucleotide sequence of SEQ ID NO:1.
[0096] As mentioned above, the present disclosure also provides a DNA sequence for expression
in a cell of a plant that, upon expression of the DNA to RNA and ingestion by a target
pest achieves suppression of a target gene in a cell, tissue or organ of an insect
pest. The dsRNA at least comprises one or multiple structural gene sequences, wherein
each of the structural gene sequences comprises a sense nucleotide sequence and an
antisense nucleotide sequence connected by a spacer sequence that forms a loop within
the complementary and antisense sequences. The sense nucleotide sequence or the antisense
nucleotide sequence is substantially identical to the nucleotide sequence of the target
gene, derivative thereof, or sequence complementary thereto. The one or more structural
gene sequences is placed operably under the control of one or more promoter sequences,
at least one of which is operable in the cell, tissue or organ of a prokaryotic or
eukaryotic organism, particularly a plant.
[0097] A gene sequence or fragment for pest control according to the present disclosure
may be cloned between two tissue specific promoters, such as two root specific promoters
which are operable in a transgenic plant cell and therein expressed to produce rnRNA
in the transgenic plant cell that form dsRNA molecules thereto. The dsRNA molecules
contained in plant tissues are ingested by an insect so that the intended suppression
of the target gene expression is achieved.
[0098] A nucleotide sequence provided by the present disclosure may comprise an inverted
repeat separated by a "spacer sequence." The spacer sequence may be a region comprising
any sequence of nucleotides that facilitates secondary structure formation between
each repeat, where this is required. In one embodiment of the present disclosure,
the spacer sequence is part of the sense or antisense coding sequence for mRNA. The
spacer sequence may alternatively comprise any combination of nucleotides or homologues
thereof that are capable of being linked covalently to a nucleic acid molecule. The
spacer sequence may comprise a sequence of nucleotides of at least about 10-100 nucleotides
in length, or alternatively at least about 100-200 nucleotides in length, at least
200-400 about nucleotides in length, or at least about 400-500 nucleotides in length.
[0099] The nucleic acid molecules or fragment of the nucleic acid molecules or other nucleic
acid molecules in the sequence listing are capable of specifically hybridizing to
other nucleic acid molecules under certain circumstances. As used herein, two nucleic
acid molecules are said to be capable of specifically hybridizing to one another if
the two molecules are capable of forming an anti-parallel, double-stranded nucleic
acid structure. A nucleic acid molecule is said to be the complement of another nucleic
acid molecule if they exhibit complete complementarity. Two molecules are said to
be "minimally complementary" if they can hybridize to one another with sufficient
stability to permit them to remain annealed to one another under at least conventional
"low-stringency" conditions. Similarly, the molecules are said to be complementary
if they can hybridize to one another with sufficient stability to permit them to remain
annealed to one another under conventional "high-stringency" conditions. Conventional
stringency conditions are described by Sambrook, et al. (1989), and by Haymes et al.
(1985).
[0100] Departures from complete complementarity are therefore permissible, as long as such
departures do not completely preclude the capacity of the molecules to form a double-
stranded structure. Thus, in order for a nucleic acid molecule or a fragment of the
nucleic acid molecule to serve as a primer or probe it needs only be sufficiently
complementary in sequence to be able to form a stable double-stranded structure under
the particular solvent and salt concentrations employed.
[0101] Appropriate stringency conditions which promote DNA hybridization are, for example,
6.0 x sodium chloride/sodium citrate (SSC) at about 45 °C, followed by a wash of 2.0
x SSC at 50 °C, are known to those skilled in the art or can be found in Current Protocols
in Molecular Biology (1989). For example, the salt concentration in the wash step
can be selected from a low stringency of about 2.0 x SSC at 50 °C to a high stringency
of about 0.2 x SSC at 50 °C. In addition, the temperature in the wash step can be
increased from low stringency conditions at room temperature, about 22°C, to high
stringency conditions at about 65 °C. Both temperature and salt may be varied, or
either the temperature or the salt concentration may be held constant while the other
variable is changed. A nucleic acid for use in the present disclosure may specifically
hybridize to one or more of nucleic acid molecules from WCR or complements thereof
under such conditions. Preferably, a nucleic acid for use in the present disclosure
will exhibit at least from about 85%, or at least from about 90%, or at least from
about 95%, or at least from about 98% or even about 100% sequence identity with a
nucleic acid molecule of SEQ ID NO:1.
[0102] Nucleic acids of the present disclosure may also be synthesized, either completely
or in part, especially where it is desirable to provide plant-preferred sequences,
by methods known in the art. Thus, all or a portion of the nucleic acids of the present
disclosure may be synthesized using codons preferred by a selected host. Species-preferred
codons may be determined, for example, from the codons used most frequently in the
proteins expressed in a particular host species. Other modifications of the nucleotide
sequences may result in mutants having slightly altered activity. dsRNA or siRNA nucleotide
sequences comprise double strands of polymerized ribonucleotide and may include modifications
to either the phosphate-sugar backbone or the nucleoside. Modifications in RNA structure
may be tailored to allow specific genetic inhibition. In one embodiment, the dsRNA
molecules may be modified through an enzymatic process so that siRNA molecules may
be generated. The siRNA can efficiently mediate the down-regulation effect for some
target genes in some insects. This enzymatic process may be accomplished by utilizing
an RNAse III enzyme or a DICER enzyme, present in the cells of an insect, a vertebrate
animal, a fungus or a plant in the eukaryotic RNAi pathway (Elbashir et al, 2002;
Hamilton and Baulcombe, 1999). This process may also utilize a recombinant DICER or
RNAse III introduced into the cells of a target insect through recombinant DNA techniques
that are readily known to the skilled in the art. Both the DICER enzyme and RNAse
III, being naturally occurring in an insect or being made through recombinant DNA
techniques, cleave larger dsRNA strands into smaller oligonucleotides. The DICER enzymes
specifically cut the dsRNA molecules into siRNA pieces each of which is about 19-25
nucleotides in length while the RNAse III enzymes normally cleave the dsRNA molecules
into 12-15 base-pair siRNA. The siRNA molecules produced by the either of the enzymes
have 2 to 3 nucleotide 3' overhangs, and 5' phosphate and 3' hydroxyl termini. The
siRNA molecules generated by RNAse III enzyme are the same as those produced by Dicer
enzymes in the eukaryotic RNAi pathway and are hence then targeted and degraded by
an inherent cellular RNA-degrading mechanism after they are subsequently unwound,
separated into single-stranded RNA and hybridize with the RNA sequences transcribed
by the target gene. This process results in the effective degradation or removal of
the RNA sequence encoded by the nucleotide sequence of the target gene in the insect.
The outcome is the silencing of a particularly targeted nucleotide sequence within
the insect. Detailed descriptions of enzymatic processes can be found in Harmon (2002).
[0103] In some embodiments, the present disclosure pertains to double stranded ribonucleotide
sequences produced from the expression of a polynucleotide according to the present
disclosure, wherein ingestion of said ribonucleotide sequence or fragments thereof
as RNAi inducing compounds by a Hemiptera crop plant pest reduces feeding of said
pest. In an advantageous embodiment, said double stranded ribonucleotide sequence
comprises a nucleic acid sequence of SEQ ID NO:2, or homologs thereof, wherein said
homologs have a sequence identity of at least 80 %, in particular of at least 85%,
in particular of at least 90%, in particular of at least 95% to SEQ ID NO: 2.
E. Incorporation of SHP Inhibitor
[0104] The manner for incorporation of the SHP inhibitor is not particularly limited, and
may be selected according to the target pest. When the target pest is a pest that
attacks a plant, for example, the agent (pesticide) containing the SHP inhibitor is
in advance retained in the plant, which is to be attacked by the target pest, through
application, spraying, or atomization. As a result of this, when the target pest ingests
the plant, the SHP inhibitor is incorporated into the body of the target pest.
[0105] On the other hand, when a feed (feed agent) containing the SHP inhibitor is placed
at the site of occurrence or in the route of entry of the target pest, the target
pest ingests the feed, and thus the SHP inhibitor is incorporated into the body of
the target pest. In addition, when the plant to be attacked is modified by the introduction
of a gene coding the SHP inhibitor, the SHP inhibitor is incorporated into the body
of the target pest when the pest ingests the transgenic plant. The transgenic plant
used in this method may be a plant subjected to gene modification so as to express:
(A) an siRNA targeted at a gene coding the SHP of the target pest; (B) an antisense
nucleic acid targeted at the transcript product of a gene coding the SHP of the target
pest; or (C) a ribozyme targeted at the transcript product of a gene coding the SHP
of the target pest.
[0106] Therefore, in some embodiments, the pest control method according to the present
disclosure comprise making a plant, which is to be attacked by the target pest, possess
an agent containing the inhibitor by application, spraying, or atomization in advance,
and incorporating the inhibitor into the body of the target pest by ingestion of the
plant.
[0107] However, in some advantageous embodiments, the pest control method according to the
present disclosure comprises incorporating the inhibitor into the body of the target
pest by ingestion of a transgenic plant containing a gene encoding the inhibitor.
E. Vectors and Host Cell Transformation
[0108] As mentioned above, the present disclosure contemplates transformation of a nucleotide
sequence of the present disclosure into a plant to achieve pest inhibitory levels
of expression of one or more dsRNA molecules. A transformation vector can be readily
prepared using methods available in the art. The transformation vector comprises one
or more nucleotide sequences that is/are capable of being transcribed to an RNA molecule
and that is/are substantially homologous and/or complementary to one or more nucleotide
sequences encoded by the genome of the insect, such that upon uptake of the RNA there
is down-regulation of expression of at least one of the respective nucleotide sequences
of the genome of the insect.
[0109] The transformation vector may be termed a dsDNA construct and may also be defined
as a recombinant molecule, an insect control agent, a genetic molecule or a chimeric
genetic construct. A chimeric genetic construct of the present disclosure may comprise,
for example, nucleotide sequences encoding one or more antisense transcripts, one
or more sense transcripts, one or more of each of the aforementioned, wherein all
or part of a transcript therefrom is homologous to all or part of an RNA molecule
comprising an RNA sequence encoded by a nucleotide sequence within the genome of an
insect.
[0110] In one embodiment the disclosure transformation vector comprises an isolated and
purified DNA molecule comprising a promoter operatively linked to one or more nucleotide
sequences of the present disclosure. The nucleotide sequence is for example SEQ ID
NO:1 or SEQ ID NO:2 or parts thereof. The nucleotide sequence includes a segment coding
all or part of a RNA present within a targeted pest RNA transcript and may comprise
inverted repeats of all or a part of a targeted pest RNA. The DNA molecule comprising
the expression vector may also contain a functional intron sequence positioned either
upstream of the coding sequence or even within the coding sequence, and may also contain
a five prime (5') untranslated leader sequence (i.e., a UTR or 5'-UTR) positioned
between the promoter and the point of translation initiation.
[0111] A plant transformation vector may contain sequences from more than one gene, thus
allowing production of more than one dsRNA for inhibiting expression of two or more
genes in cells of a target pest. One skilled in the art will readily appreciate that
segments of DNA whose sequence corresponds to that present in different genes can
be combined into a single composite DNA segment for expression in a transgenic plant.
Alternatively, a plasmid of the present disclosure already containing at least one
DNA segment can be modified by the sequential insertion of additional DNA segments
between the enhancer and promoter and terminator sequences. In the insect control
agent of the present disclosure designed for the inhibition of multiple genes, the
genes to be inhibited can be obtained from the same insect species in order to enhance
the effectiveness of the insect control agent. In certain embodiments, the genes can
be derived from different insects in order to broaden the range of insects against
which the agent is effective. When multiple genes are targeted for suppression or
a combination of expression and suppression, a polycistronic DNA element can be fabricated
as illustrated and disclosed in Fillatti, Application Publication No.
US 2004-0029283.
[0112] Promoters that function in different plant species are also well known in the art.
Promoters useful for expression of polypeptides in plants include those that are inducible,
viral, synthetic, or constitutive as described in Odell et al. (1985), and/or promoters
that are temporally regulated, spatially regulated, and spatio-temporally regulated.
Preferred promoters include the enhanced CaMV35S promoters, the SUC2 promotor and
the FMV35S promoter. For the purpose of the present disclosure, e.g., for optimum
control of species that feed from the phloem via their stylet, it may be preferable
to achieve the highest levels of expression of these genes within the phloems of plants.
Therefore, in an advantageous embodiment the promotor is active in the phloem of a
crop plant like the CaMV 35S promotor (Yang and Christou, 1990) and the SUC2 promoter
(Truermit and Sauer, 1995). dsRNA expression control by the CaMV 35S promoter was
used by Pitino et al. (2011) that demonstrated host induced gene silencing (HIGS)
in aphids.
[0113] The phloem located expression of target specific dsRNA or siRNA in genetically modified
plants that targets SHP allows most likely the reduction of infestation of crop plants
by aphids and other plant sucking insects of the groups Sternorryhncha and Fulgoromorpha
under the critical economic threshold, which is declared aim of IPS. In this context
varying length of dsRNA and siRNA are possible that cover different regions of SHP
mRNA.
[0114] A recombinant DNA vector or construct of the present disclosure will typically comprise
a selectable marker that confers a selectable phenotype on plant cells. Selectable
markers may also be used to select for plants or plant cells that contain the exogenous
nucleic acids encoding polypeptides or proteins of the present disclosure. The marker
may encode biocide resistance, antibiotic resistance (e.g., kanamycin, G418 bleomycin,
hygromycin, etc.), or herbicide resistance (e.g., glyphosate, etc.). Examples of selectable
markers include, but are not limited to, a neo gene which codes for kanamycin resistance
and can be selected for using kanamycin, G418, etc., a bar gene which codes for bialaphos
resistance; a mutant EPSP synthase gene which encodes glyphosate resistance; a nitrilase
gene which confers resistance to bromoxynil; a mutant acetolactate synthase gene (ALS)
which confers imidazolinone or sulfonylurea resistance; and a methotrexate resistant
DHFR gene. Examples of such selectable markers are illustrated in
U.S. Patents 5,550,318;
5,633,435;
5,780,708 and
6,118,047.
[0115] A recombinant vector or construct of the present disclosure may also include a screenable
marker. Screenable markers may be used to monitor expression. Exemplary screenable
markers include a [beta]-glucuronidase or uidA gene (GUS) which encodes an enzyme
for which various chromogenic substrates are known (Jefferson, 1987; Jefferson et
al, 1987); an R-locus gene, which encodes a product that regulates the production
of anthocyanin pigments (red color) in plant tissues (Dellaporta et al, 1988); a [beta]-lactamase
gene (Sutcliffe et al, 1978), a gene which encodes an enzyme for which various chromogenic
substrates are known (e.g., PADAC, a chromogenic cephalosporin); a luciferase gene
(Ow et al, 1986) a xylE gene (Zukowsky et al, 1983) which encodes a catechol dioxygenase
that can convert chromogenic catechols; an [alpha]-amylase gene (Bcatu et al, 1990);
a tyrosinase gene (Katz et al, 1983) which encodes an enzyme capable of oxidizing
tyrosine to DOPA and dopaquinone which in turn condenses to melanin; an α -galactosidase,
which catalyzes a chromogenic α- galactose substrate.
[0116] In some advantageous embodiments, the isolated polynucleotides according to the present
disclosure are operably linked to a heterologous promoter and/or are defined as comprised
on a plant transformation vector.
[0117] Preferred plant transformation vectors include those derived from a Ti plasmid of
Agrobacterium tumefaciens (e.g. U.S. Patent Nos. 4,536,475, 4,693,977, 4,886,937, 5,501,967 and
EP 0 122 791). Agrobacterium rhizogenes plasmids (or "Ri") are also useful and known in the art.
Other preferred plant transformation vectors include those disclosed, e.g., by Herrera-Estrella
(1983); Bevan (1983), Klee (1985) and
EP 0 120 516. In an advantageous embodiment, the vector is a binary vector.
[0118] In general it is preferred to introduce a functional recombinant DNA at a non-specific
location in a plant genome. In special cases it may be useful to insert a recombinant
DNA construct by site-specific integration. Several site-specific recombination systems
exist which are known to function implants include cre-lox as disclosed in
U.S. Patent 4,959,317 and FLP-FRT as disclosed in
U.S. Patent 5,527,695.
[0119] Suitable methods for transformation of host cells for use with the current plant
are believed to include virtually any method by which DNA can be introduced into a
cell, such as by direct delivery of DNA such as by PEG-mediated transformation of
protoplasts (Omirulleh et al, 1993), by desiccation/inhibition-mediated DNA uptake
(Potrykus et al, 1985), by electroporation (
U.S. Patent No. 5,384,253), by agitation with silicon carbide fibers (Kaeppler et al, 1990;
U.S. Patent No. 5,302,523; and
U.S. Patent No. 5,464,765), by Agrobacterium-mediated transformation (
U.S. Patent No. 5,591,616 and
U.S. Patent No. 5,563,055) and by acceleration of DNA coated particles (
U.S. Patent No. 5,550,318;
U.S. Patent No. 5,538,877; and
U.S. Patent No. 5,538,880), etc. Through the application of techniques such as these, the cells of virtually
any species may be stably transformed. In the case of multicellular species, the transgenic
cells may be regenerated into transgenic organisms. Methods for the creation of transgenic
plants and expression of heterologous nucleic acids in plants in particular are known
and may be used with the nucleic acids provided herein to prepare transgenic plants
that exhibit reduced susceptibility to feeding by a target pest organism such as corn
rootworms. Plant transformation vectors can be prepared, for example, by inserting
the dsRNA producing nucleic acids disclosed herein into plant transformation vectors
and introducing these into plants. One known vector system has been derived by modifying
the natural gene transfer system of
Agrobacterium tumefaciens. The natural system comprises large Ti (tumor-inducing)-plasmids containing a large
segment, known as T-DNA, which is transferred to transformed plants. Another segment
of the Ti plasmid, the vir region, is responsible for T-DNA transfer. The T-DNA region
is bordered by terminal repeats, hi the modified binary vectors the tumor-inducing
genes have been deleted and the functions of the vir region are utilized to transfer
foreign DNA bordered by the T-DNA border sequences. The T-region may also contain
a selectable marker for efficient recovery of transgenic plants and cells, and a multiple
cloning site for inserting sequences for transfer such as a dsRNA encoding nucleic
acid.
[0120] A transgenic plant formed using Agrobacterium transformation methods typically contains
a single simple recombinant DNA sequence inserted into one chromosome and is referred
to as a transgenic event. Such transgenic plants can be referred to as being heterozygous
for the inserted exogenous sequence. A homozygous transgenic plant can be obtained
by sexually mating (selfmg) an independent segregant transgenic plant to produce FI
seed. One fourth of the FI seed produced will be homozygous with respect to the transgene.
Germinating FI seed results in plants that can be tested for heterozygosity or homozygosity,
typically using a SNP assay or a thermal amplification assay that allows for the distinction
between heterozygotes and homozygotes (i.e., a zygosity assay).
[0121] The methods and compositions of the present disclosure may be applied to any monocot
and dicot plant, depending on the coleopteran pest control desired. Specifically,
the plants are intended to include, without limitation, alfalfa, aneth, apple, apricot,
artichoke, arugula, asparagus, avocado, banana, barley, beans, beet, blackberry, blueberry,
broccoli, brussel sprouts, cabbage, canola, cantaloupe, carrot, cassava, cauliflower,
celery, cherry, cilantro, citrus, Clementine, coffee, corn, cotton, cucumber, Douglas
fir, eggplant, endive, escarole, eucalyptus, fennel, figs, gourd, grape, grapefruit,
honey dew, jicama, kiwifruit, lettuce, leeks, lemon, lime, Loblolly pine, mango, melon,
mushroom, nut, oat, okra, onion, orange, an ornamental plant, papaya, parsley, pea,
peach, peanut, pear, pepper, persimmon, pine, pineapple, plantain, plum, pomegranate,
poplar, potato, pumpkin, quince, radiata pine, radicchio, radish, raspberry, rice,
rye, sorghum, Southern pine, soybean, spinach, squash, strawberry, sugarbeet, sugarcane,
sunflower, sweet potato, sweetgum, tangerine, tea, tobacco, tomato, turf, a vine,
watermelon, wheat, yams, and zucchini plants. Thus, a plant transformed with a recombinant
DNA sequence of SEQ ID NO:1, or concatemer, fragment, or complement thereof, that
is transcribed to produce at least one dsRNA molecule that functions when ingested
by a coleopteran pest to inhibit the expression of a target gene in the pest is also
provided by the plant. In particular embodiments, the recombinant DNA sequence is
SEQ ID NO:2, or fragments, complements, or concatemers thereof.
[0122] However, the polynucleotide according to the present disclosure may be transformed,
transduced or transfected via a recombinant DNA vector also in a prokaryotic cell
or eukaryotic cell, for example for production of an agent (pesticide) containing
the SHP inhibitor.
[0123] A recombinant DNA vector may, for example, be a linear or a closed circular plasmid.
The vector system may be a single vector or plasmid or two or more vectors or plasmids
that together contain the total DNA to be introduced into the genome of the bacterial
host. In addition, a bacterial vector may be an expression vector. The nucleic acid
molecules according to the present disclosure can, for example, be suitably inserted
into a vector under the control of a suitable promoter that functions in one or more
microbial hosts to drive expression of a linked coding sequence or other DNA sequence.
Many vectors are available for this purpose, and selection of the appropriate vector
will depend mainly on the size of the nucleic acid to be inserted into the vector
and the particular host cell to be transformed with the vector. Each vector contains
various components depending on its function (amplification of DNA or expression of
DNA) and the particular host cell with which it is compatible. The vector components
for bacterial transformation generally include, but are not limited to, one or more
of the following: a signal sequence, an origin of replication, one or more selectable
marker genes, and an inducible promoter allowing the expression of exogenous DNA.
F. Target Gene Suppression
[0124] The present disclosure provides, as an example, a transformed host or symbiont pest
target organism, transformed plant cells and transformed plants and their progeny.
The transformed plant cells and transformed plants may be engineered to express one
or more of the dsRNA or siRNA sequences described herein to provide a pest-protective
effect. These sequences may be used for SHP gene suppression in a SHP expressing pest
organism, thereby reducing the predation by the pest on a protected transformed host
or symbiont organism. As used herein the words "gene suppression" are intended to
refer to any of the well-known methods for reducing the levels of gene transcription
to mRNA and/or subsequent translation of the mRNA.
[0125] Gene suppression is also intended to mean the reduction of protein expression from
a gene or a coding sequence including posttranscriptional gene suppression and transcriptional
suppression. Posttranscriptional gene suppression is mediated by the homology between
of all or a part of a mRNA transcribed from a gene or coding sequence targeted for
suppression and the corresponding double stranded RNA used for suppression, and refers
to the substantial and measurable reduction of the amount of available mRNA available
in the cell for binding by ribosomes. The transcribed RNA can be in the sense orientation
to effect what is called co-suppression, in the anti-sense orientation to effect what
is called anti-sense suppression, or in both orientations producing a dsRNA to effect
what is called RNA interference (RNAi).
[0126] Transcriptional suppression is mediated by the presence in the cell of a dsRNA gene
suppression agent exhibiting substantial sequence identity to a promoter DNA sequence
or the complement thereof to effect what is referred to as promoter trans suppression.
Gene suppression may be effective against a native plant gene associated with a trait,
e.g., to provide plants with reduced levels of a protein encoded by the native gene
or with enhanced or reduced levels of an affected metabolite. Gene suppression can
also be effective against target genes in plant pests that may ingest or contact plant
material containing gene suppression agents, specifically designed to inhibit or suppress
the expression of one or more homologous or complementary sequences in the cells of
the pest. Post-transcriptional gene suppression by anti-sense or sense oriented RNA
to regulate gene expression in plant cells is disclosed in U.S. Pat. Nos. 5,107,065,
5,759,829, 5,283,184, and 5,231,020. The use of dsRNA to suppress genes in plants
is disclosed in
WO 99/53050,
WO 99/49029, U.S. Patent Application Publication No. 2003/0175965, and 2003/0061626,
U.S. Patent Application No.10/465,800, and
U.S. Patent Nos. 6,506,559, and
6,326,193.
[0127] A beneficial method of post transcriptional gene suppression in plants employs both
sense-oriented and anti-sense-oriented, transcribed RNA which is stabilized, e.g.,
as a hairpin and stem and loop structure. A preferred DNA construct for effecting
post transcriptional gene suppression is one in which a first segment encodes an RNA
exhibiting an anti-sense orientation exhibiting substantial identity to a segment
of a gene targeted for suppression, which is linked to a second segment in sense orientation
encoding an RNA exhibiting substantial complementarity to the first segment. Such
a construct forms a stem and loop structure by hybridization of the first segment
with the second segment and a loop structure from the nucleotide sequences linking
the two segments (see
WO94/01550 WO98/05770, US 2002/0048814, and US 2003/0018993).
[0128] According to one embodiment of the present disclosure, there is provided a nucleotide
sequence, for which
in vitro expression results in transcription of a stabilized RNA sequence that is substantially
homologous to an RNA molecule of a targeted gene in an insect that comprises an RNA
sequence encoded by a nucleotide sequence within the genome of the insect. Thus, after
the insect ingests the stabilized RNA sequence incorporated in a diet or sprayed on
a plant surface, a down-regulation of the nucleotide sequence corresponding to the
target gene in the cells of a target insect is affected.
[0129] Inhibition of the SHP target gene using the stabilized dsRNA technology of the present
disclosure is sequence-specific in that nucleotide sequences corresponding to the
duplex region of the RNA are targeted for genetic inhibition. RNA containing a nucleotide
sequences identical to a portion of the target gene is preferred for inhibition. RNA
sequences with insertions, deletions, and single point mutations relative to the target
sequence have also been found to be effective for inhibition. In performance of the
present disclosure, it is preferred that the inhibitory dsRNA and the portion of the
target gene share at least from about 80% sequence identity, or from about 90% sequence
identity, or from about 95% sequence identity, or from about 99% sequence identity,
or even about 100% sequence identity. Alternatively, the duplex region of the RNA
may be defined functionally as a nucleotide sequence that is capable of hybridizing
with a portion of the target gene transcript. A less than MI length sequence exhibiting
a greater homology compensates for a longer less homologous sequence. The length of
the identical nucleotide sequences may be at least about 25, 50, 100, 200, 300, 400,
500 or at least about 1000 bases. Normally, a sequence of greater than 20-100 nucleotides
should be used, though a sequence of greater than about 200-300 nucleotides would
be preferred, and a sequence of greater than about 500-1000 nucleotides would be especially
preferred depending on the size of the target gene. The plant has the advantage of
being able to tolerate sequence variations that might be expected due to genetic mutation,
strain polymorphism, or evolutionary divergence. The introduced nucleic acid molecule
may not need to be absolute homology, may not need to be full length, relative to
either the primary transcription product or fully processed mRNA of the target gene.
Therefore, those skilled in the art need to realize that, as disclosed herein, 100%
sequence identity between the RNA and the target gene is not required to practice
the present disclosure. Inhibition of target gene expression may be quantified by
measuring either the endogenous target RNA or the protein produced by translation
of the target RNA and the consequences of inhibition can be confirmed by examination
of the outward properties of the cell or organism. Techniques for quantifying RNA
and proteins are well known to one of ordinary skill in the art. Multiple selectable
markers are available that confer resistance to ampicillin, bleomycin, chloramphenicol,
gentamycin, hygromycin, kanamycin, lincomycin, methotrexate, phosphinothricin, puromycin,
spectinomycin, rifampicin, and tetracyclin, and the like.
[0130] In certain embodiments gene expression is inhibited by at least 10%, preferably by
at least 33%, more preferably by at least 50%, and yet more preferably by at least
80%. In particularly preferred embodiments of the plant gene expression is inhibited
by at least 80%, more preferably by at least 90%, more preferably by at least 95%,
or by at least 99% within cells in the insect so a significant inhibition takes place.
Significant inhibition is intended to refer to sufficient inhibition that results
in a detectable phenotype (e.g., cessation of larval growth, paralysis or mortality,
etc.) or a detectable decrease in RNA and/or protein corresponding to the target gene
being inhibited. Although in certain embodiments of the disclosure inhibition occurs
in substantially all cells of the insect, in other preferred embodiments inhibition
occurs in only a subset of cells expressing the gene. For example, if the gene to
be inhibited plays an essential role in cells in the insect alimentary tract, inhibition
of the gene within these cells is sufficient to exert a deleterious effect on the
insect. dsRNA molecules may be synthesized either in vivo or in vitro. The dsRNA may
be formed by a single self-complementary RNA strand or from two complementary RNA
strands. Endogenous RNA polymerase of the cell may mediate transcription in vivo,
or cloned RNA polymerase can be used for transcription in vivo or in vitro. Inhibition
may be targeted by specific transcription in an organ, tissue, or cell type; stimulation
of an environmental condition (e.g., infection, stress, temperature, chemical inducers);
and/or engineering transcription at a developmental stage or age. The RNA strands
may or may not be polyadenylated; the RNA strands may or may not be capable of being
translated into a polypeptide by a cell's translational apparatus.
[0131] A RNA, dsRNA, siRNA, or miRNA of the present disclosure may be produced chemically
or enzymatically by one skilled in the art through manual or automated reactions or
in vivo in another organism. RNA may also be produced by partial or total organic
synthesis; any modified ribonucleotide can be introduced by in vitro enzymatic or
organic synthesis. The RNA may be synthesized by a cellular RNA polymerase or a bacteriophage
RNA polymerase (e.g., T3, T7, SP6). The use and production of an expression construct
are known in the art (see, for example,
WO 97/32016; U.S. Pat. No's. 5,593, 874, 5,698,425, 5,712,135, 5,789,214, and 5,804,693). If
synthesized chemically or by in vitro enzymatic synthesis, the RNA may be purified
prior to introduction into the cell. For example, RNA can be purified from a mixture
by extraction with a solvent or resin, precipitation, electrophoresis, chromatography,
or a combination thereof. Alternatively, the RNA may be used with no or a minimum
of purification to avoid losses due to sample processing. The RNA may be dried for
storage or dissolved in an aqueous solution. The solution may contain buffers or salts
to promote annealing, and/or stabilization of the duplex strands.
[0132] For transcription from a transgene
in vivo or an expression construct, a regulatory region (e.g., promoter, enhancer, silencer,
and polyadenylation) may be used to transcribe the RNA strand (or strands). Therefore,
in one embodiment, the nucleotide sequences for use in producing RNA molecules may
be operably linked to one or more promoter sequences functional in a microorganism,
a fungus or a plant host cell. Ideally, the nucleotide sequences are placed under
the control of an endogenous promoter, normally resident in the host genome. The nucleotide
sequence of the present disclosure, under the control of an operably linked promoter
sequence, may further be flanked by additional sequences that advantageously affect
its transcription and/or the stability of a resulting transcript. Such sequences are
generally located upstream of the operably linked promoter and/or downstream of the
3' end of the expression construct and may occur both upstream of the promoter and
downstream of the 3' end of the expression construct, although such an upstream sequence
only is also contemplated.
[0133] The present disclosure provides for inhibiting gene expression of one or multiple
target genes in a target pest using stabilized dsRNA methods. The plant is particularly
useful in the modulation of eukaryotic gene expression, in particular the modulation
of expression of genes present in pests that exhibit a digestive system pH level that
is from about 4.5 to about 9.5, more preferably from about 5.0 to about 8.0, and even
more preferably from about 6.5 to about 7.5. For plant pests with a digestive system
that exhibits pH levels outside of these ranges, delivery methods may be desired for
use that do not require ingestion of dsRNA molecules.
[0134] The modulatory effect of dsRNA is applicable to a variety of genes expressed in the
pests including, for example, endogenous genes responsible for cellular metabolism
or cellular transformation, including house-keeping genes, transcription factors and
other genes which encode polypeptides involved in cellular metabolism.
[0135] The present disclosure provides in part a delivery system for the delivery of the
insect control agents to insects through their exposure to a diet containing the insect
control agents of the present disclosure. In accordance with one of the embodiments,
the stabilized dsRNA or siRNA molecules may be incorporated in the insect diet or
may be overlaid on the top of the diet for consumption by an insect. The present disclosure
also provides in part a delivery system for the delivery of the insect control agents
to insects through their exposure to a microorganism or host such as a plant containing
the insect control agents of the present disclosure by ingestion of the microorganism
or the host cells or the contents of the cells. In accordance with another embodiment,
the present disclosure involves generating a transgenic plant cell or a plant that
contains a recombinant DNA construct transcribing the stabilized dsRNA molecules of
the present disclosure. As used herein, the phrase "generating a transgenic plant
cell or a plant" refers to the methods of employing the recombinant DNA technologies
readily available in the art (e.g., by Sambrook, et al, 1989) to construct a plant
transformation vector transcribing the stabilized dsRNA molecules of the present disclosure,
to transform the plant cell or the plant and to generate the transgenic plant cell
or the transgenic plant that contain the transcribed, stabilized dsRNA molecules.
[0136] In still another embodiment, non-pathogenic, attenuated strains of microorganisms
may be used as a carrier for the insect control agents and, in this perspective, the
microorganisms carrying such agents are also referred to as insect control agents.
The microorganisms may be engineered to express a nucleotide sequence of a target
gene to produce RNA molecules comprising RNA sequences homologous or complementary
to RNA sequences typically found within the cells of an insect. Exposure of the insects
to the microorganisms result in ingestion of the microorganisms and down-regulation
of expression of target genes mediated directly or indirectly by the RNA molecules
or fragments or derivatives thereof.
[0137] The present disclosure alternatively provides exposure of an insect to the insect
control agents of the present disclosure incorporated in a spray mixer and applied
to the surface of a host, such as a host disclosure, hi an exemplary embodiment, ingestion
of the insect control agents by an insect delivers the insect control agents to the
gut of the insect and subsequently to the cells within the body of the insect. In
another embodiment, infection of the insect by the insect control agents through other
means such as by injection or other physical methods also permits delivery of the
insect control agents. In yet another embodiment, the RNA molecules themselves are
encapsulated in a synthetic matrix such as a polymer and applied to the surface of
a host such as a plant. Ingestion of the host cells by an insect permits delivery
of the insect control agents to the insect and results in down-regulation of a target
gene in the host.
[0138] It is envisioned that the compositions of the present disclosure can be incorporated
within the seeds of a plant species either as a product of expression from a recombinant
gene incorporated into a genome of the plant cells, or incorporated into a coating
or seed treatment that is applied to the seed before planting. The plant cell containing
a recombinant gene is considered herein to be a transgenic event. It is believed that
a pesticidal seed treatment can provide significant advantages when combined with
a transgenic event that provides protection from coleopteran pest infestation that
is within the preferred effectiveness range against a target pest. In addition, it
is believed that there are situations that are well known to those having skill in
the art, where it is advantageous to have such transgenic events within the preferred
range of effectiveness.
[0139] The present disclosure provides in part a delivery system for the delivery of insect
control agents to insects. The stabilized dsRNA or siRNA molecules of the present
disclosure may be directly introduced into the cells of an insect, or introduced into
an extracellular cavity, interstitial space, lymph system, digestive system, into
the circulation of the insect through oral ingestion or other means that one skilled
in the art may employ. Methods for oral introduction may include direct mixing of
RNA with food of the insect, as well as engineered approaches in which a species that
is used as food is engineered to express the dsRNA or siRNA, then fed to the insect
to be affected. In one embodiment, for example, the dsRNA or siRNA molecules may be
incorporated into, or overlaid on the top of, the insect's diet. In another embodiment,
the RNA may be sprayed onto a plant surface. In still another embodiment, the dsRNA
or siRNA may be expressed by microorganisms and the microorganisms may be applied
onto a plant surface or introduced into a root, stem by a physical means such as an
injection. In still another embodiment, a plant may be genetically engineered to express
the dsRNA or siRNA in an amount sufficient to kill the insects known to infect the
plant.
[0140] Specifically, in practicing the present disclosure in WCR, the stabilized dsRNA or
siRNA may be introduced in the midgut inside the insect and achieve the desired inhibition
of the targeted genes. The dsRNA or siRNA molecules may be incorporated into a diet
or be overlaid on the diet as discussed above and may be ingested by the insects.
In any event, the dsRNA's of the present disclosure are provided in the diet of the
target pest. The target pest of the present disclosure will exhibit a digestive tract
pH from about 4.5 to about 9.5, or from about 5 to about 8.5, or from about 6 to about
8, or from about 6.5 to about 7.7, or about 7.0. The digestive tract of a target pest
is defined herein as the location within the pest that food that is ingested by the
target pest is exposed to an environment that is favorable for the uptake of the dsRNA
molecules of the present disclosure without suffering a pH so extreme that the hydrogen
bonding between the double-strands of the dsRNA are caused to dissociate and form
single stranded molecules.
[0141] It is also anticipated that dsRNA's produced by chemical or enzymatic synthesis may
be formulated in a manner consistent with common agricultural practices and used as
spray-on products for controlling insect infestations. The formulations may include
the appropriate stickers and wetters required for efficient foliar coverage as well
as UV protectants to protect dsRNAs from UV damage. Such additives are commonly used
in the bio-insecticide industry and are well known to those skilled in the art. Such
applications could be combined with other spray-on insecticide applications, biologically
based or not, to enhance plant protection from insect feeding damage.
[0142] The present inventors contemplate that bacterial strain producing insecticidal proteins
may be used to produce dsRNAs for insect control purposes. These strains may exhibit
improved insect control properties. A variety of different bacterial hosts may be
used to produce insect control dsRNAs. Exemplary bacteria may include
E. coli, B. thuringiensis, Pseudomonas sp., Photorhabdus sp., Xenorhabdus sp., Serratia
entomophila and related Serratia sp., B. sphaericus, B. cereus, B. laterosporus, B.
popilliae, Clostridium bifermentans and other Clostridium species, or other spore-forming gram-positive bacteria. In
certain embodiments, bacteria may be engineered for control of pests such as mosquitoes.
[0143] The present plant also relates to recombinant DNA constructs for expression in a
microorganism. Exogenous nucleic acids from which a RNA of interest is transcribed
can be introduced into a microbial host cell, such as a bacterial cell or a fungal
cell, using methods known in the art.
[0144] The nucleotide sequences of the present disclosure may be introduced into a wide
variety of prokaryotic and eukaryotic microorganism hosts to produce the stabilized
dsRNA or siRNA molecules.
F. Transgenic Plants
[0145] Another aspect of the present disclosure relates to a transgenic plant and seeds.
A gene coding an inhibitor against the SHP of the target pest has been introduced
into the transgenic plant of the present disclosure. Typically, the transgenic plant
of the present disclosure has been subjected to gene modification so as to express:
(A) a dsRNA molecule, wherein the dsRNA may be modified in
the plant through an enzymatic process so that siRNA molecules may be generated targeting a transcript product of a gene coding the SHP of the target pest ; (B)
an antisense nucleic acid targeted at the transcript product of a gene coding the
SHP of the target pest; or (C) a ribozyme targeted at the transcript product of a
gene coding the SHP of the target pest.
[0146] As mentioned above, the present disclosure provides seeds and plants having one or
more transgenic event. Combinations of events are referred to as "stacked" transgenic
events. These stacked transgenic events can be events that are directed at the same
target pest, or they can be directed at different target pests. In one embodiment,
a seed having the ability to express a nucleic acid provided herein also has the ability
to express at least one other insecticidal agent, including, but not limited to, an
RNA molecule the sequence of which is derived from the sequence of an RNA expressed
in a target pest and that forms a double stranded RNA structure upon expressing in
the seed or cells of a plant grown from the seed, wherein the ingestion of one or
more cells or the cell content of the plant by the target pest results in the suppression
of expression of the RNA in the cells of the target pest. In further embodiments,
a seed having the ability to express a dsRNA the sequence of which is derived from
a target pest also has a transgenic event that provides herbicide tolerance. One beneficial
example of a herbicide tolerance gene provides resistance to glyphosate, N-(phosphonomethyl)
glycine, including the isopropylamine salt form of such herbicide.
[0147] In the present method, combination of expression of an insecticidal amount of a dsRNA
within the cells of a transgenic seed or plant grown from the seed coupled with treatment
of the seed or plant with certain chemical or protein pesticides may be used to provide
unexpected synergistic advantages, including unexpectedly superior efficacy for protection
against damage to the resulting transgenic plant by the target pest. In particular
embodiments, treatment of a transgenic seed that is capable of expressing certain
constructs that form dsRNA molecules, the sequence of which are derived from one or
more sequences expressed in a corn rootworm, with from about 100 gm to about 400 gm
of pesticide per 100 kg of seed provides unexpectedly superior protection against
corn rootworm. In addition, it is believed that such combinations are also effective
to protect the emergent plants against predation by other pests. The seeds of the
present disclosure may also be used to decrease the cost of pesticide use, because
less pesticide can be used to obtain a required amount of protection than when such
methods are not used. Moreover, because less pesticide is used and because it is applied
prior to planting and without a separate field application, it is believed that the
subject method is therefore safer to the operator and to the environment, and is potentially
less expensive than conventional methods.
[0148] Pesticides and insecticides that are useful in compositions in combination with the
methods and compositions of the present disclosure, including as seed treatments and
coatings as well as methods for using such compositions can be found, for example,
in
U.S. Patent 6,551,962, the entirety of which is incorporated herein by reference.
[0149] It is anticipated that the combination of certain stabilized dsRNA constructs with
one or more insect control protein genes will result in synergies that enhance the
insect control phenotype of a transgenic plant. Insect bioassays employing artificial
diet- or whole plant tissue can be used to define dose-responses for larval mortality
or growth inhibition using both dsRNAs and insect control proteins. One skilled in
the art can test mixtures of dsRNA molecules and insect control proteins in bioassay
to identify combinations of actives that are synergistic and desirable for deployment
in insect-protected plants (Tabashnik, 1992). Synergy in killing insect pests has
been reported between different insect control proteins (for review, see Schnepf et
al., 1998). It is anticipated that synergies will exist between certain dsRNAs and
between certain dsRNAs and certain insect control proteins.
[0150] The disclosure also relates to commodity products containing one or more of the sequences
of the present disclosure, and produced from a recombinant plant or seed containing
one or more of the nucleotide sequences of the present disclosure are specifically
contemplated as embodiments of the present disclosure. A commodity product containing
one or more of the sequences of the present disclosure is intended to include, but
not be limited to, meals, oils, crushed or whole grains or seeds of a plant, or any
food product comprising any meal, oil, or crushed or whole grain of a recombinant
plant or seed containing one or more of the sequences of the present disclosure. The
detection of one or more of the sequences of the present disclosure in one or more
commodity or commodity products contemplated herein is defacto evidence that the commodity
or commodity product is composed of a transgenic plant designed to express one or
more of the nucleotide.
H. Obtaining Nucleic Acids
[0151] Some embodiments pertain to isolated and purified nucleotide sequences as SHP inhibitors
that may be used as the insect control agents.
[0152] Therefore, the present disclosure provides a method for obtaining a nucleic acid
comprising a nucleotide sequence for producing a dsRNA or siRNA. In one embodiment,
such a method for obtaining a nucleic acid fragment comprises a nucleotide sequence
for producing a substantial portion of a dsRNA or siRNA comprises: (a) synthesizing
first and a second oligonucleotide primers corresponding to a portion of one of the
nucleotide sequences from a targeted insect; and (b) amplifying a cDNA or gDNA template
in a cloning vector using the first and second oligonucleotide primers of step (a)
wherein the amplified nucleic acid molecule transcribes a substantial portion of a
dsRNA or siRNA of the present invention. The preferred target genes of the present
disclosure are genes encoding SHP.
[0153] In one embodiment, a gene is selected that is expressed in the insect gut. Targeting
genes expressed in the gut avoids the requirement for the dsRNA to spread within the
insect. Target genes for use in the present invention may include, for example, those
that share substantial homologies to the nucleotide sequences of known gut-expressed
genes that encode protein components of the vacuolar and plasma membrane proton V-ATPase
(Dow et al., 1997; Dow, 1999). This protein complex is the sole energizer of epithelial
ion transport and is responsible for alkalinization of the midgut lumen. The V-ATPase
is also expressed in the Malpighian tubule, an outgrowth of the insect hindgut that
functions in fluid balance and detoxification of foreign compounds in a manner analogous
to a kidney organ of a mammal. In another embodiment, the V-ATPase may be Vha68-2,
or a homolog or ortholog thereof (e.g. as found in SEQ ID NO:821).
[0154] For the purpose of the present invention, the dsRNA or siRNA molecules may be obtained
from a SHP encoding DNA or RNA by polymerase chain (PCR) amplification of a target
SHP gene sequences.
[0155] Nucleic acid molecules and fragments thereof from amphids, or other Hemiptera pest
species may be employed to obtain other nucleic acid molecules from other species
for use in the present disclosure to produce desired dsRNA and siRNA molecules. Such
nucleic acid molecules include the nucleic acid molecules that encode the complete
coding sequence of a protein and promoters and flanking sequences of such molecules.
In addition, such nucleic acid molecules include nucleic acid molecules that encode
for gene family members. Such molecules can be readily obtained by using the above-described
nucleic acid molecules or fragments thereof to screen, for instance, cDNA or gDNA
libraries. Methods for forming such libraries are well known in the art.
[0156] In order to obtain a DNA segment from the corresponding SHP gene in an insect species,
PCR primers may be designed based on the sequence as found in the insects from which
the SHP gene has been cloned. The primers are designed to amplify a DNA segment of
sufficient length for use in the present disclosure. DNA (either genomic DNA or cDNA)
is prepared from the insect species, and the PCR primers are used to amplify the DNA
segment. Amplification conditions are selected so that amplification will occur even
if the primers do not exactly match the target sequence. Alternately, the gene (or
a portion thereof) may be cloned from a gDNA or cDNA library prepared from the insect
pest species, using the SHP gene or another known insect gene as a probe. Techniques
for performing PCR and cloning from libraries are known. Further details of the process
by which DNA segments from target insect pest species may be isolated based on the
sequence of the SHP genes previously cloned from
Acyrtosiphon pisum or other insect species are provided in the Examples. One of ordinary skill in the
art will recognize that a variety of techniques may be used to isolate gene segments
from insect pest species that correspond to genes previously isolated from other species.
[0157] The described agro-biotechnological approach of HIGS of SHP in crops (e.g. wheat,
cotton, beans, potatoe and tomato), where plant sucking insects of the groups Sternorryhncha
and Fulgoromorpha are relevant pests on, can be used to control these in the field
as well as in the greenhouse. The development of resistances by pests, observed many
times by varies insects (mentioned above), can be excluded on the current state of
knowledge. Off-target effects on other insects can actually be excluded because no
hits were detected by BLAST search in mRNA sequences of available organisms (Carolan
et al., 2009).
I. Figures
[0158]
Figure 1 shows the Influence of SHP silencing on sheath formation. Salivary sheaths
from untreated aphids reared on an artificial diet (a, b) show a typical necklace
structure and the sheaths are wider at the stylet penetration site (white arrow) than
at the tip. Each bead represents one gel saliva secretion event (white arrowheads).
Aphids injected with IMPI dsRNA form similar sheaths (c, d). The hole caused by stylet
penetration through the Parafilm sheet is visible (white arrows). SHP silencing disrupts
sheath formation (e-h). In aphids injected with 25 ng dsRNA (e, f) the first two beads
are clear and the next 4-5 appear less distinct. Additional gel saliva material appears
to be distributed over the surrounding Parafilm sheet surface. In aphids injected
with 50 ng dsRNA there are no visible beads (g, h) and only a small amount of gel
saliva material covering the hole in the sheet (white arrow).
Figure 2 shows the temporal evolution of behavior (waveforms) SHP RNAi aphids and
controls. The percentage of individuals in the control group (a) and the SHP RNAi
group (b) is shown demonstrating specific behaviors at 30 min intervals over a total
recording time of 8 hours.
Figure 3 shows a comparison of most relevant aphid behavior. In comparison with control
injected aphids (IMPI), show silenced aphids show a higher percentage of stylet movement
(C) and reduced ingestion (E2). Secretion of watery saliva (E1) does not differ.
Figure 4 shows the reproduction SHP RNAi aphids and controls. Each group contained
15 aphids. (a) The SHP RNAi aphids show a lower reproduction rate and a shorter overall
duration of reproduction than untreated and IMPI RNAi controls. (b) The total reproduction
of the SHP RNAi aphids is significantly lower than that of the control groups.
Figure 5 shows the survival analysis of SHP RNAi aphids and controls by Kaplan Meier
Log-Rank. Aphids that died for unrelated reasons are censored (black circles). Each
group contained 15 aphids and the experiment was carried out three times.
[0159] The following methods and examples are offered for illustrative purposes only, and
are not intended to limit the scope of the present disclosure in any way.
Methods and Examples
[0160] In the following examples, materials and methods of the present disclosure are provided
including the determination of the effect of SHP silencing on pest reproduction. It
should be understood that these examples are for illustrative purpose only and are
not to be construed as limiting this disclosure in any manner. All publications, patents,
and patent applications cited herein are hereby incorporated by reference in their
entirety for all purposes.
Example 1: Aphid and plant breeding
[0161] The
Acyrtosiphon pisum clone LL01 was reared on 2-3-week-old bean plants (Vicia faba var. minor) in a climate
cabinet (KBWF 720, Binder GmbH, Tuttlingen, Germany) with a 16-h photoperiod and a
day/night temperature of 24/18°C. Plants for experiments and aphid rearing were cultivated
in a greenhouse with an average temperature of 20°C and natural light plus additional
illumination (SONT Agro 400W, Phillips, Eindhoven, Netherlands) to maintain a 14-h
photoperiod.
Example 2: dsRNA production and injection
[0162] A 491-bp template for the production of dsRNA representing the
A. pisum SHP sequence (ACYPI009881) was generated by PCR from plasmid DNA using gene-specific
primers containing a 5' T7 polymerase promoter sequence (AP-SHP-for 5'-TAA TAC GAC
TCA CTA TAG GGA GAC GTT ATT ATT GCT GCT GCT GTG-3' and AP-SHP-back 5'-TAA TAC GAC
TCA CTA TAG GGA GAA CAG CTA CCC TGG CCG ATC TT-3'). The sequence was ensured that
it did not have overlaps exceeding 19 bp with any other gene, to avoid off-target
effects. The template was purified using the QIAquick PCR Purification Kit (Qiagen,
Hilden, Germany) and dsRNA was prepared using the Ambion MEGAscript RNAi kit (Applied
Biosystems, Austin, TX). The primers were designed with Primer3 (Rozen S, Skaletsky
HJ (2000) and were purchased from Sigma-Aldrich (Taufkirchen, Germany). dsRNA was
used representing the
Galleria mellonella insect metalloproteinase inhibitor gene (AY330624) as a control (Wedde M, et al.,
2007).
15 nl of dsRNA solution was injected under a stereomicroscope by using a Nanoliter
2000 injector together with a Sys-Micro4 controller (World Precision Instruments,
Berlin, Germany). Glass microcapillaries for injection were pulled with a PN-30 puller
(Narishige International Limited, London, UK). Prior to injection, aphids were immobilized
with their dorsal thorax on a vacuum holder (van Helden M, Tjallingii WF, 2000). The
dsRNA was injected at a rate of 2 nl/s between the mesothorax and methathorax, as
previously described (Mutti NS, Park Y, Reese JC, Reeck GR, 2006).
Example 3: Rearing aphids during experimental treatments
[0163] Aphids were reared on detached, mature
V. faba leaves cut from intact plants with a razor blade. Petiole section of 1-5 mm in length
was cut again under water and the leaf was transferred to a Petri dish, filled to
a height of 7 mm with 1.5% tap water agar (Carl-Roth GmbH, Karlsruhe, Germany) containing
0.03% methyl-4-hydroxybenzoate (Sigma-Aldrich). Leafs were inserted into the cooled
agar upside down and the Petri dishes were maintained in a climate cabinet as described
above. Senescent leafs were replaced.
Example 4: Preparation of aphid salivary sheaths and observation by scanning electron
microscopy
[0164] Aphids were reared on an artificial diet that mimics the cell-wall milieu (20 mM
KCI, 1 mM CaCl2, 10 mM MES, adjusted to pH 5.5 (Will T, et al., 2012; Cosgrove DJ,
Cleland RE, 1983) to enforce secretion of gel saliva. The food was sterile-filtered
before use (pore size 0.45 µm) and 150 µl was placed between two Parafilm sheets (sachet),
previously sterilized with 30% H2O2 for at least 30 min. Five days after dsRNA injection
15 aphids of each treatment were placed in groups of five per sheet. The sachet was
located on one side of a plastic ring. Opposite to the diet sachet, the ring was closed
with a single Parafilm sheet after the ring volume was filled with water. The diet
sachet was then placed downwards on a small aphid cage and aphids were allowed to
feed for 24 h. Sheets containing aphids were then placed downwards in a Petri dish
and were searched for salivary sheaths with an inverse microscope (Olympus IMT-2).
Regions of interest were labeled, SEM sample holders were placed on these regions
and Parafilm was cut around the sample holders with a scalpel. The samples were dried
for a minimum of 3 days in a desiccator with silica gel under vacuum, then gold-sputtered
and observed with a Zeiss DSM982 Gemini SEM. Two replicas were prepared for each treatment
and 20 randomly-chosen salivary sheaths were observed for each replica.
[0165] As a result, the formation of the aphid salivary sheath was disrupted by SHP silencing.
The Aphids (
Acyrthosiphon pisum) were injected with 25 ng of double-stranded RNA (dsRNA) corresponding to the major
salivary sheath protein (SHP) and compared to non-treated controls and non-relevant
dsRNA controls (injected with 25 ng of dsRNA corresponding to the insect metalloprotease
inhibitor IMPI from the greater wax moth
Galleria mellonella (Clermont A, et al., 2004) which is absent from aphids) when fed on artificial diets
through Parafilm. After 5 days, salivary sheaths were prepared for scanning electron
microscopy (SEM). This revealed that salivary sheaths secreted by the control aphids
adopted the typical necklace-like structure that forms on this substrate (Fig. 1A-D,
white arrows), whereas those secreted by the SHP RNAi aphids showed the remnants of
a bead-like structure but were predominantly amorphous (Fig. 1E and F). The injection
of 50 ng of dsRNA almost completely abolished any bead-like structures, with minimal
gel saliva deposits observed at the stylet penetration sites (Fig. 1G and H, white
arrows). The silencing of shp mRNA was confirmed by quantitative real time PCR (data
not shown).
Example 5: EPG analysis of aphid feeding behavior
[0166] Aphids injected with 50 ng of dsRNA were selected for further structural and behavioral
analysis. Aphid feeding behavior was monitored using the electrical penetration graph
(EPG) technique (Tjallingii WF, 1988). A gold wire electrode (1 cm x 20 µm) was attached
to the dorsal abdomen of randomly selected apterous aphids 5 days after injection,
using electrically conductive silver glue (Electrolube, Swadlincote, Derbyshire, UK)
and a vacuum device for immobilization (van Helden M, Tjallingii WF, 2000). The aphid
electrode was connected to a DC EPG Giga-8 (Tjallingii WF, 1988, Tjallingii WF, 1978)
and the EPG output was recorded with Stylet+ (hardware and software from EPG Systems,
Wageningen, Netherlands). A second electrode (plant electrode) was inserted into the
soil of potted plants. The experimental setup was placed in a Farraday cage to shield
it from electromagnetic interference. Aphids were placed on the lower side of the
petiole of a mature leaf on a 10-day-old plant, and EPG recordings were started immediately,
running for 8 h. 14 biological replicates were carried out for each treatment. EPG
waveforms were analyzed by pattern and autopower spectra (Prado E, Tjallingii WF,
1994) using the Stylet+ analysis module. Further analysis was performed with the workbook
for automatic parameter calculation of EPG data version 4.4 (Sarria E, et al., 2009).
[0167] The possibility that SHP silencing could affect interactions with the epidermis,
mesophyll and phloem was considered and we therefore analyzed 37 of the 132 calculated
parameters listed in the workbook for automatic parameter calculation, electrical
penetration graph (EPG) data version 4.4 (Table S1). As a result the SHP silencing
increases aphid probing activity but delays and inhibits feeding.
[0168] In Table 1, data from selected parameters of Table S1 were sorted as events in classes
representing 2 h intervals and analyzed using the non-parametric Wald-Wolfowitz-test.
Table 1: Non-parametric analysis of phloem localizing-parameters in SHP RNAi aphids
and controls
|
Start of EPG to 1st sustained E2 |
1st probe to 1st sustained E2 |
Time |
IMPI |
SHP |
IMPI |
SHP |
0-2h |
4 |
5 |
4 |
5 |
2-4h |
8 |
1 |
8 |
1 |
4-6h |
0 |
4 |
0 |
4 |
6-8h |
2 |
1 |
2 |
1 |
no detection |
0 |
3 |
0 |
3 |
Z (corr.) |
2.1184 |
2.1184 |
P |
0.0208 |
0.0341 |
[0169] Results indicate clearly that sustained E2 (successful long-term access to a sieve
tube (nutrition source) is significantly delayed as a consequence of
shp silencing. In addition, interrupted sheath formation results in a higher percentage
of non-phloem behavior over the complete observation time of 8 hours (Fig. 2).
[0170] The results in Table 2 indicate that SHP silenced aphids show a higher percentage
of stylet movement in the plant (
C) and a reduced ingestion (
E2). The secretion of watery saliva after sieve tube penetration (
E1) is not influenced. Data of grey filled cells (Table 2) are displayed in Figure 3.
The percentage of sustained ingestion events is reduced for SHP silenced aphids.
Example 6: Survival and reproduction assay
[0171] Survival assays (n = 3) and reproduction assays (n = 1) were conducted separately
using 15 aphids per group in each test. Aphids were maintained on a single leaf in
an agar plate as described above. Parameters were checked once every day from the
first day after injection until the final aphid died. Plates were placed in a climate
cabinet using the conditions described above.
[0172] As a result it could be shown that SHP silencing inhibits aphid reproduction. The
reproduction of aphids was monitored in SHP RNAi group and control groups for the
lifespan of selected aphids. In all groups, the reproduction rate increased rapidly
at the beginning of the observation period and reached a maximum after 4 days (Fig.
4a). The maximum reproduction rate in the control groups was approximately eight nymphs
per day, whereas in the SHP RNAi group it was six nymphs per day. Furthermore, reproduction
in the control groups was maintained for 27 days (non-treated control) or 22 days
(IMPI RNAi control) whereas the reproduction rate dropped off after 4 days in the
SHP RNAi group and ceased after 17 days. There was a highly significant difference
(
p < 0.001) in total mean reproduction (Fig. 4b) between the SHP RNAi group (45.6 nymphs
per adult) and untreated controls (88.2 nymphs per adult), and a slight significant
difference (
p = 0.052) between the SHP RNAi group and IMPI RNAi group (68.9 nymphs per adult).
There was no significant difference between the two control groups (
p = 0.083).
[0173] In view to survival, no differences were observed between the three different groups,
non-treated, dsRNA IMPI injected and shp silenced (Fig. 5).
Example 7: Quantitative real time PCR
[0174] RNA was isolated from aphids 5 days after injection of dsRNA IMPI and dsRNA SHP respectively,
3x 10 aphids for each treatment, as previously described by using TriReagent (Sigma-Aldrich)
and was immediately stored at -80°C. mRNA was converted to cDNA (First Strand cDNA
Synthesis Kit; Fermentas, St. Leon-Rot, Germany) after a cleanup (RNeasy MiniElute
Cleanup Kit; Quiagen, Hilden, Germany) and subsequent qPCR was performed with the
CFX96 real time PCR detection system (Bio-Rad, Hercules, CA, USA) using SensiMix SYBR
No-ROX One-Step Kit (Bioline, Luckenwalde, Germany). Appropriate primers were designed
using Primer3 (18) (AP-SHP-qPCR-for 5'-AAA TGT TGC GTT GTG GAC TT-3' and AP-SHP-qPCR-back
5'-GGT AAT CCT TGA AGG GGA GA-3') and were purchased from Sigma-Aldrich. The amplified
sequence was different to the one used for dsRNA production.
Example 8: Statistical analysis
[0175] Descriptive statistical analysis of aphid behavior was performed with Origin 8.1G
(OriginLab Corporation, Northampton, MA, USA) while comparison of treatments was performed
with ANOVA and ANOVA on ranks using SigmaPlot 11 (Systat Software Inc., London, UK).
The Wald-Wolfowitz test (SigmaPlot 11) was used to analyze non-parametric class-arranged
behavior data. Because of the small sample size for non-parametric data analysis,
Z and p values were corrected (Siegel S, 1956). Survival analysis was performed with
Kaplan-Meier Survival Analysis Log-Rank (SigmaPlot 11), and ANOVA was used to compare
the median and maximum survival rates. Reproduction data were analyzed by ANOVA. The
level for significance for the statistical tests was set to p = 0.05, whereas p-values
between 0.05 and 0.075 indicated a trend with marginal significance.
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